Neprilysin inhibitors

ABSTRACT

In one aspect, the invention relates to compounds having the formula: 
                         
where R 1 , R 2a , R 2b , R 3 —R 6 , a, b, Z, and X are as defined in the specification, or a pharmaceutically acceptable salt thereof. These compounds have neprilysin inhibition activity. In another aspect, the invention relates to pharmaceutical compositions comprising such compounds; methods of using such compounds; and processes and intermediates for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/869,174, filed Apr.24, 2013, now U.S. Pat. No. 8,846,913; which is a divisional of U.S.Ser. No. 13/398,027, filed Feb. 16, 2012, now U.S. Pat. No. 8,449,890;which claims the benefit of U.S. Provisional Application No. 61/443,828,filed on Feb. 17, 2011; the entire disclosures of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel compounds havingneprilysin-inhibition activity. The invention also relates topharmaceutical compositions comprising such compounds, processes andintermediates for preparing such compounds and methods of using suchcompounds to treat diseases such as hypertension, heart failure,pulmonary hypertension, and renal disease.

2. State of the Art

Neprilysin (neutral endopeptidase, EC 3.4.24.11) (NEP), is anendothelial membrane bound Zn²⁺ metallopeptidase found in many organsand tissues, including the brain, kidneys, lungs, gastrointestinaltract, heart, and the peripheral vasculature. NEP degrades andinactivates a number of endogenous peptides, such as enkephalins,circulating bradykinin, angiotensin peptides, and natriuretic peptides,the latter of which have several effects including, for example,vasodilation and natriuresis/diuresis, as well as inhibition of cardiachypertrophy and ventricular fibrosis. Thus, NEP plays an important rolein blood pressure homeostasis and cardiovascular health.

NEP inhibitors, such as thiorphan, candoxatril, and candoxatrilat, havebeen studied as potential therapeutics. Compounds that inhibit both NEPand angiotensin-I converting enzyme (ACE) are also known, and includeomapatrilat, gempatrilat, and sampatrilat. Referred to as vasopeptidaseinhibitors, this latter class of compounds is described in Robl et al.(1999) Exp. Opin. Ther. Patents 9(12): 1665-1677.

SUMMARY OF THE INVENTION

The present invention provides novel compounds that have been found topossess neprilysin (NEP) enzyme inhibition activity. Accordingly,compounds of the invention are expected to be useful and advantageous astherapeutic agents for treating conditions such as hypertension andheart failure.

One aspect of the invention relates to a compound of formula I:

where:

R¹ is selected from —OR⁷ and —NR⁸R⁹;

R^(2a) is selected from —OH, —OP(O)(OH)₂, and —OC(O)CH(R³⁷)NH₂, andR^(2b) is —CH₃; or R^(2a) and R^(2b) are both —CH₃; or R^(2a) and R^(2b)are taken together to form —CH₂—CH₂—; or R^(2a) is taken together withR⁷ to form —OCR¹⁸R¹⁹— or is taken together with R⁸ to form —OC(O)—;

Z is selected from —CH— and —N—;

X is a —C₁₋₉heteroaryl;

R³ is absent or is selected from H; halo; —C₀₋₅alkylene-OH; —NH₂;—C₁₋₆alkyl; —CF₃; —C₃₋₇cycloalkyl; —C₀₋₂alkylene-O—C₁₋₆alkyl; —C(O)R²⁰;—C₀₋₁alkylene-COOR²¹; —C(O)NR²²R²³; —NHC(O)R²⁴; ═O; —NO₂; —C(CH₃)═N(OH);phenyl optionally substituted with one or two groups independentlyselected from halo, —OH, —CF₃, —OCH₃, —NHC(O)CH₃, and phenyl;naphthalenyl; pyridinyl; pyrazinyl; pyrazolyl optionally substitutedwith methyl; thiophenyl optionally substituted with methyl or halo;furanyl; and —CH₂-morpholinyl; and R³, when present, is attached to acarbon atom;

R⁴ is absent or is selected from H; —OH; —C₁₋₆alkyl;—C₁₋₂alkylene-COOR³⁵; —CH₂OC(O)CH(R³⁶)NH₂; —OCH₂OC(O)CH(R³⁶)NH₂;—OCH₂OC(O)CH₃; —CH₂OP(O)(OH)₂; —CH₂CH(OH)CH₂OH;—CH[CH(CH₃)₂]—NHC(O)O—C₁₋₆alkyl; pyridinyl; and phenyl or benzyloptionally substituted with one or more groups selected from halo,—COOR³⁵, —OCH₃, —OCF₃, and —SCF₃; and R⁴, when present, is attached to acarbon or nitrogen atom;

or R³ and R⁴ are taken together to form -phenylene-O—(CH₂)₁₋₃— or-phenylene-O—CH₂—CHOH—CH₂—;

-   -   a is 0 or 1; R⁵ is selected from halo, CH₃, CF₃, and —CN;    -   b is 0 or an integer from 1 to 3; each R⁶ is independently        selected from halo, —OH, —CH₃, OCH₃, and —CF₃;

R⁷ is selected from H; —C₁₋₈alkyl; —C₁₋₃alkylene-C₆₋₁₀aryl;—C₁₋₃alkylene-C₁₋₉heteroaryl; —C₃₋₇cycloalkyl; —[(CH₂)₂O]₁₋₃CH₃;—C₁₋₆alkylene-OC(O)R¹⁰; —C₁₋₆alkylene-NR¹²R¹³, —C₁₋₆alkylene-C(O)R³¹,—C₀₋₆alkylenemorpholinyl; —C₁₋₆alkylene-SO₂—C₁₋₆alkyl;

R¹⁰ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹²R¹³, —CH[CH(CH₃)₂]—NH₂,—CH[CH(CH₃)₂]—NHC(O)O—C₁₋₆alkyl, and —CH(NH₂)CH₂COOCH₃; and R¹² and R¹³are independently selected from H, —C₁₋₆alkyl, and benzyl, or R¹² andR¹³ are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—;R³¹ is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR¹²R¹³; and R³² is—C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl;

R⁸ is selected from H, —OH, —OC(O)R¹⁴, —CH₂COOH, —O-benzyl, pyridyl, and—OC(S)NR¹⁵R¹⁶; R¹⁴ is selected from H, —C₁₋₆alkyl, —C₆₋₁₀aryl,—OCH₂—C₆₋₁₀aryl, —CH₂O—C₆₋₁₀aryl, and —NR¹⁵R¹⁶; and R¹⁵ and R¹⁶ areindependently selected from H and —C₁₋₄alkyl;

R⁹ is selected from H, —C₁₋₆alkyl, and —C(O)R¹⁷; and R¹⁷ is selectedfrom H, —C₁₋₆alkyl, —C₃₋₇cycloalkyl, —C₆₋₁₀aryl, and —C₁₋₉heteroaryl;

R¹⁸ and R¹⁹ are independently selected from H, —C₁₋₆alkyl, and—O—C₃₋₇cycloalkyl, or R¹⁸ and R¹⁹are taken together to form ═O;

R²⁰ is selected from H and —C₁₋₆alkyl;

R²¹ and R³⁵ are independently selected from H, —C₁₋₆alkyl,—C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl,—[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R²⁵, —C₁₋₆alkylene-NR²⁷R²⁸,—C₁₋₆alkylene-C(O)R³³, —C₀₋₆alkylenemorpholinyl,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

R²⁵ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR²⁷R²⁸, —CH[CH(CH₃)₂]—NH₂,—CH[CH(CH₃)₂]—NHC(O)O—C₁₋₆alkyl, and —CH(NH₂)CH₂COOCH₃; and R²⁷ and R²⁸are independently selected from H, —C₁₋₆alkyl, and benzyl, or R²⁷ andR²⁸ are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—;R³³ is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR²⁷R²⁸; and R³⁴ is—C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl;

R²² and R²³ are independently selected from H, —C₁₋₆alkyl, —CH₂COOH,—(CH₂)₂OH; —(CH₂)₂OCH₃, —(CH₂)₂SO₂NH₂, —(CH₂)₂N(CH₃)₂,—C₀₋₁alkylene-C₃₋₇cycloalkyl, and —(CH₂)₂-imidazolyl; or R²² and R²³ aretaken together to form a saturated or partially unsaturated—C₃₋₅heterocycle optionally substituted with halo, —OH, —COOH, or—CONH₂; and optionally containing an oxygen atom in the ring;

R²⁴ is selected from —C₁₋₆alkyl; —C₀₋₁alkylene-O—C₁₋₆alkyl; phenyloptionally substituted with halo or —OCH₃; and —C₁₋₉heteroaryl; and

R³⁶ is selected from H, —CH(CH₃)₂, phenyl, and benzyl; and

R³⁷ is selected from H, —CH(CH₃)₂, phenyl, and benzyl;

where each alkyl group in R¹, R³, and R⁴ is optionally substituted with1 to 8 fluoro atoms; and;

where the methylene linker on the biphenyl is optionally substitutedwith one or two —C₁₋₆alkyl groups or cyclopropyl;

or a pharmaceutically acceptable salt thereof.

Another aspect of the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a compound of theinvention. Such compositions may optionally contain other therapeuticagents. Accordingly, in yet another aspect of the invention, apharmaceutical composition comprises a compound of the invention as thefirst therapeutic agent, one or more secondary therapeutic agent, and apharmaceutically acceptable carrier. Another aspect of the inventionrelates to a combination of active agents, comprising a compound of theinvention and a second therapeutic agent. The compound of the inventioncan be formulated together or separately from the additional agent(s).When formulated separately, a pharmaceutically acceptable carrier may beincluded with the additional agent(s). Thus, yet another aspect of theinvention relates to a combination of pharmaceutical compositions, thecombination comprising: a first pharmaceutical composition comprising acompound of the invention and a first pharmaceutically acceptablecarrier; and a second pharmaceutical composition comprising a secondtherapeutic agent and a second pharmaceutically acceptable carrier. Inanother aspect, the invention relates to a kit containing suchpharmaceutical compositions, for example where the first and secondpharmaceutical compositions are separate pharmaceutical compositions.

Compounds of the invention possess NEP enzyme inhibition activity, andare therefore expected to be useful as therapeutic agents for treatingpatients suffering from a disease or disorder that is treated byinhibiting the NEP enzyme or by increasing the levels of its peptidesubstrates. Thus, one aspect of the invention relates to a method oftreating patients suffering from a disease or disorder that is treatedby inhibiting the NEP enzyme, comprising administering to a patient atherapeutically effective amount of a compound of the invention. Anotheraspect of the invention relates to a method of treating hypertension,heart failure, or renal disease, comprising administering to a patient atherapeutically effective amount of a compound of the invention. Stillanother aspect of the invention relates to a method for inhibiting a NEPenzyme in a mammal comprising administering to the mammal, a NEPenzyme-inhibiting amount of a compound of the invention.

Since compounds of the invention possess NEP inhibition activity, theyare also useful as research tools. Accordingly, one aspect of theinvention relates to a method of using a compound of the invention as aresearch tool, the method comprising conducting a biological assay usinga compound of the invention. Compounds of the invention can also be usedto evaluate new chemical compounds. Thus another aspect of the inventionrelates to a method of evaluating a test compound in a biological assay,comprising: (a) conducting a biological assay with a test compound toprovide a first assay value; (b) conducting the biological assay with acompound of the invention to provide a second assay value; wherein step(a) is conducted either before, after or concurrently with step (b); and(c) comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include a NEP enzymeinhibition assay. Still another aspect of the invention relates to amethod of studying a biological system or sample comprising a NEPenzyme, the method comprising: (a) contacting the biological system orsample with a compound of the invention; and (b) determining the effectscaused by the compound on the biological system or sample.

Yet another aspect of the invention relates to processes andintermediates useful for preparing compounds of the invention.Accordingly, another aspect of the invention relates to a process ofpreparing compounds of formula I, comprising the step of coupling acompound of formula 1 with a compound of formula 2:

to produce a compound of formula I; where P¹ is H or an amino-protectinggroup selected from t-butoxycarbonyl, trityl, benzyloxycarbonyl,9-fluorenylmethoxycarbonyl, formyl, trimethylsilyl, andt-butyldimethylsilyl; and where the process further comprisesdeprotecting the compound of formula 1 when P¹ is an amino protectinggroup; and where R¹, R^(2a), R^(2b), R³—R⁶, a, b, Z, and X are asdefined for formula I. Another aspect of the invention relates to aprocess of preparing a pharmaceutically acceptable salt of a compound offormula I, comprising contacting a compound of formula I in free acid orbase form with a pharmaceutically acceptable base or acid. In otheraspects, the invention relates to products prepared by any of theprocesses described herein, as well as novel intermediates used in suchprocess. In one aspect of the invention novel intermediates have formula1, 9, 10, 11, 12, or a salt thereof, as defined herein.

Yet another aspect of the invention relates to the use of a compound offormula I or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament, especially for the manufacture of amedicament useful for treating hypertension, heart failure, or renaldisease. Another aspect of the invention relates to use of a compound ofthe invention for inhibiting a NEP enzyme in a mammal Still anotheraspect of the invention relates to the use of a compound of theinvention as a research tool. Other aspects and embodiments of theinvention are disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an,” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, and so forth used herein are tobe understood as being modified in all instances by the term “about,”unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each number should at least be construed in lightof the reported significant digits and by applying ordinary roundingtechniques.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms and include, for example,—C₁₋₄alkyl, —C₁₋₅alkyl, —C₂₋₅alkyl, —C₁₋₆alkyl, —C₁₋₈alkyl, and—C₁₋₁₀alkyl. Representative alkyl groups include, by way of example,methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl,n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown preceding the term assubscript. For example, the term “—C₁₋₆alkyl” means an alkyl grouphaving from 1 to 6 carbon atoms, and the term “—C₃₋₇cycloalkyl” means acycloalkyl group having from 3 to 7 carbon atoms, respectively, wherethe carbon atoms are in any acceptable configuration.

The term “alkylene” means a divalent saturated hydrocarbon group thatmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 0 to 10 carbon atoms and include, forexample, —C₀₋₁alkylene-, —C₀₋₆alkylene-, —C₁₋₃alkylene-, and—C₁₋₆alkylene-. Representative alkylene groups include, by way ofexample, methylene, ethane-1,2-diyl (“ethylene”), propane-1,2-diyl,propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl and the like. It isunderstood that when the alkylene term include zero carbons such as—C₀₋₁alkylene-, such terms are intended to include the absence of carbonatoms, that is, the alkylene group is not present except for a covalentbond attaching the groups separated by the alkylene term.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or one or more fused rings. Fused ring systemsinclude those that are fully unsaturated (e.g., naphthalene) as well asthose that are partially unsaturated (e.g.,1,2,3,4-tetrahydronaphthalene). Unless otherwise defined, such arylgroups typically contain from 6 to 10 carbon ring atoms and include, forexample, —C₆₋₁₀aryl. Representative aryl groups include, by way ofexample, phenyl and naphthalene-1-yl, naphthalene-2-yl, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms and include, for example,—C₃₋₅cycloalkyl, —C₃₋₆cycloalkyl and —C₃₋₇cycloalkyl. Representativecycloalkyl groups include, by way of example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heterocycle” is intended to include monovalent unsaturated(aromatic) heterocycles having a single ring or two fused rings as wellas monovalent saturated and partially unsaturated groups having a singlering or multiple condensed rings. The heterocycle ring can contain from3 to 15 total ring atoms, of which 1 to 14 are ring carbon atoms, and 1to 4 are ring heteroatoms selected from nitrogen, oxygen or sulfur.Typically, however, the heterocycle ring contains from 3 to 10 totalring atoms, of which 1 to 9 are ring carbon atoms, and 1 to 4 are ringheteroatoms. The point of attachment is at any available carbon ornitrogen ring atom. Exemplary heterocycles include, for example,—C₁₋₇heterocycle, —C₃₋₅heterocycle, —C₂₋₆heterocycle, —C₃₋₁₂heterocycle,—C₅₋₉heterocycle, —C₁₋₉heterocycle, —C₁₋₁₁heterocycle, and—C₁₋₁₄heterocyle.

Monovalent unsaturated heterocycles are also commonly referred to as“heteroaryl” groups. Unless otherwise defined, heteroaryl groupstypically contain from 5 to 10 total ring atoms, of which 1 to 9 arering carbon atoms, and 1 to 4 are ring heteroatoms, and include, forexample, —C₁₋₉heteroaryl and —C₅₋₉heteroaryl. Representative heteroarylgroups include, by way of example, pyrrole (e.g., 3-pyrrolyl and2H-pyrrol-3-yl), imidazole (e.g., 2-imidazolyl), furan (e.g., 2-furyland 3-furyl), thiophene (e.g., 2-thienyl), triazole (e.g.,1,2,3-triazolyl and 1,2,4-triazolyl), pyrazole (e.g., 1H-pyrazol-3-yl),oxazole (e.g., 2-oxazolyl), isoxazole (e.g., 3-isoxazolyl), thiazole(e.g., 2-thiazolyl and 4-thiazolyl), and isothiazole (e.g.,3-isothiazolyl), pyridine (e.g., 2-pyridyl, 3-pyridyl, and 4-pyridyl),pyridylimidazole, pyridyltriazole, pyrazine, pyridazine (e.g.,3-pyridazinyl), pyrimidine (e.g., 2-pyrimidinyl), tetrazole, triazine(e.g., 1,3,5-triazinyl), indolyle (e.g., 1H-indol-2-yl, 1H-indol-4-yland 1H-indol-5-yl), benzofuran (e.g., benzofuran-5-yl), benzothiophene(e.g., benzo[b]thien-2-yl and benzo[b]thien-5-yl), benzimidazole,benzoxazole, benzothiazole, benzotriazole, quinoline (e.g., 2-quinolyl),isoquinoline, quinazoline, quinoxaline and the like.

Monovalent saturated heterocycles typically contain from 3 to 10 totalring atoms, of which 2 to 9 are ring carbon atoms, and 1 to 4 are ringheteroatoms, and include, for example —C₃₋₅heterocycle. Representativemonovalent saturated heterocycles include, by way of example, monovalentspecies of pyrrolidine, imidazolidine, pyrazolidine, piperidine,1,4-dioxane, morpholine, thiomorpholine, piperazine, 3-pyrroline and thelike. In some instances, moieties may be described as being takentogether to form a saturated —C₃₋₅heterocycle optionally containing anoxygen atom in the ring. Such groups include:

Monovalent partially unsaturated heterocycles typically contain from 3to 10 total ring atoms, of which 2 to 11 are ring carbon atoms, and 1 to3 are ring heteroatoms, and include, for example —C₃₋₅heterocycle and—C₂₋₁₂heterocycle. Representative monovalent partially unsaturatedheterocycles include, by way of example, pyran, benzopyran, benzodioxole(e.g., benzo[1,3]dioxol-5-yl), tetrahydropyridazine,2,5-dihydro-1H-pyrrole, dihydroimidazole, dihydrotriazole,dihydrooxazole, dihydroisoxazole, dihydrothiazole, dihydroisothiazole,dihydrooxadiazole, dihydrothiadiazole, tetrahydropyridazine,hexahydropyrroloquinoxaline, and dihydrooxadiazabenzo[e]azulene. In someinstances, moieties may be described as being taken together to form apartially unsaturated —C₃₋₅heterocycle. Such groups include:

The term “optionally substituted” means that group in question may beunsubstituted or it may be substituted one or several times, such as 1to 3 times, or 1 to 5 times, or 1 to 8 times. For example, a phenylgroup that is “optionally substituted” with halo atoms, may beunsubstituted, or it may contain 1, 2, 3, 4, or 5 halo atoms; and analkyl group that is “optionally substituted” with fluoro atoms may beunsubstituted, or it may contain 1, 2, 3, 4, 5, 6, 7, or 8 fluoroatoms;. Similarly, a group that is “optionally substituted” with one ortwo —C₁₋₆alkyl groups, may be unsubstituted, or it may contain one ortwo —C₁₋₆alkyl groups.

As used herein, the phrase “having the formula” or “having thestructure” is not intended to be limiting and is used in the same waythat the term “comprising” is commonly used. For example, if onestructure is depicted, it is understood that all stereoisomer andtautomer forms are encompassed, unless stated otherwise.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable manner with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug administration.

The term “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (for example, salts having acceptable mammalian safetyfor a given dosage regime). However, it is understood that the saltscovered by the invention are not required to be pharmaceuticallyacceptable salts, such as salts of intermediate compounds that are notintended for administration to a patient. Pharmaceutically acceptablesalts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. In addition, when a compound of formula I contains both a basicmoiety, such as an amine, pyridine or imidazole, and an acidic moietysuch as a carboxylic acid or tetrazole, zwitterions may be formed andare included within the term “salt” as used herein. Salts derived frompharmaceutically acceptable inorganic bases include ammonium, calcium,copper, ferric, ferrous, lithium, magnesium, manganic, manganous,potassium, sodium, and zinc salts, and the like. Salts derived frompharmaceutically acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable inorganic acids include salts of boric,carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric orhydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Saltsderived from pharmaceutically acceptable organic acids include salts ofaliphatic hydroxyl acids (for example, citric, gluconic, glycolic,lactic, lactobionic, malic, and tartaric acids), aliphaticmonocarboxylic acids (for example, acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (for example, aspartic andglutamic acids), aromatic carboxylic acids (for example, benzoic,p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylaceticacids), aromatic hydroxyl acids (for example, o-hydroxybenzoic,p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids(for example, fumaric, maleic, oxalic and succinic acids), glucoronic,mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(for example, benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, and the like.

As used herein, the term “prodrug” is intended to mean an inactive (orsignificantly less active) precursor of a drug that is converted intoits active form in the body under physiological conditions, for example,by normal metabolic processes. Such compounds may not possesspharmacological activity at NEP, but may be administered orally orparenterally and thereafter metabolized in the body to form compoundsthat are pharmacologically active at NEP. Exemplary prodrugs includeesters such as C₁₋₆alkylesters and aryl-C₁₋₆alkylesters. In oneembodiment, the active compound has a free carboxyl and the prodrug isan ester derivative thereof, i.e., the prodrug is an ester such as—C(O)OCH₂CH₃. Such ester prodrugs are then converted by solvolysis orunder physiological conditions to be the free carboxyl compound. Theterm is also intended to include certain protected derivatives ofcompounds of formula I that may be made prior to a final deprotectionstage. Thus, all protected derivatives and prodrugs of compounds formulaI are included within the scope of the invention.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need thereof, thatis, the amount of drug needed to obtain the desired therapeutic effect.For example, a therapeutically effective amount for treatinghypertension is an amount of compound needed to, for example, reduce,suppress, eliminate, or prevent the symptoms of hypertension, or totreat the underlying cause of hypertension. In one embodiment, atherapeutically effective amount is that amount of drug needed to reduceblood pressure or the amount of drug needed to maintain normal bloodpressure. On the other hand, the term “effective amount” means an amountsufficient to obtain a desired result, which may not necessarily be atherapeutic result. For example, when studying a system comprising a NEPenzyme, an “effective amount” may be the amount needed to inhibit theenzyme.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as hypertension) in apatient, such as a mammal (particularly a human) that includes one ormore of the following: (a) preventing the disease or medical conditionfrom occurring, i.e., preventing the reoccurrence of the disease ormedical condition or prophylactic treatment of a patient that ispre-disposed to the disease or medical condition; (b) ameliorating thedisease or medical condition, i.e., eliminating or causing regression ofthe disease or medical condition in a patient; (c) suppressing thedisease or medical condition, i.e., slowing or arresting the developmentof the disease or medical condition in a patient; or (d) alleviating thesymptoms of the disease or medical condition in a patient. For example,the term “treating hypertension” would include preventing hypertensionfrom occurring, ameliorating hypertension, suppressing hypertension, andalleviating the symptoms of hypertension (for example, lowering bloodpressure). The term “patient” is intended to include those mammals, suchas humans, that are in need of treatment or disease prevention or thatare presently being treated for disease prevention or treatment of aspecific disease or medical condition, as well as test subjects in whichcompounds of the invention are being evaluated or being used in anassay, for example an animal model.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

In one aspect, the invention relates to compounds of formula I:

or a pharmaceutically acceptable salt thereof.

As used herein, the term “compound of the invention” includes allcompounds encompassed by formula I such as the species embodied informulas Ia to If, Ia-1 to Ia-4, Ib-1, Ib-2, Ic-1, Ic-2, Id-1, and Id-2,as well as the compounds encompassed by formulas II-VIII, and speciesthereof. Similarly, reference to compound of a given formula is intendedto include all species; for example the term “compound of formula III”is intended to include the species IIIa and IIIb, and so forth. Inaddition, the compounds of the invention may also contain several basicor acidic groups (for example, amino or carboxyl groups) and therefore,such compounds can exist as a free base, free acid, or in various saltforms. All such salt forms are included within the scope of theinvention. Furthermore, the compounds of the invention may also exist asprodrugs. Accordingly, those skilled in the art will recognize thatreference to a compound herein, for example, reference to a “compound ofthe invention” or a “compound of formula I” includes a compound offormula I as well as pharmaceutically acceptable salts and prodrugs ofthat compound unless otherwise indicated. Further, the term “or apharmaceutically acceptable salt and/or prodrug thereof” is intended toinclude all permutations of salts and prodrugs, such as apharmaceutically acceptable salt of a prodrug. Furthermore, solvates ofcompounds of formula I are included within the scope of this invention.

The compounds of formula I may contain one or more chiral centers andtherefore, these compounds may be prepared and used in variousstereoisomeric forms. Accordingly, the invention also relates to racemicmixtures, pure stereoisomers (e.g., enantiomers and diastereoisomers),stereoisomer-enriched mixtures, and the like unless otherwise indicated.When a chemical structure is depicted herein without anystereochemistry, it is understood that all possible stereoisomers areencompassed by such structure. Thus, for example, the terms “compound offormula I,” “compounds of formula II,” and so forth, are intended toinclude all possible stereoisomers of the compound. Similarly, when aparticular stereoisomer is shown or named herein, it will be understoodby those skilled in the art that minor amounts of other stereoisomersmay be present in the compositions of the invention unless otherwiseindicated, provided that the utility of the composition as a whole isnot eliminated by the presence of such other isomers. Individualstereoisomers may be obtained by numerous methods that are well known inthe art, including chiral chromatography using a suitable chiralstationary phase or support, or by chemically converting them intodiastereoisomers, separating the diastereoisomers by conventional meanssuch as chromatography or recrystallization, then regenerating theoriginal stereoisomer.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds ofthe invention are included within the scope of the invention unlessotherwise specified. For example, if X is depicted as (R⁴ beinghydrogen):

it is understood that the compound may also exist in a tautomeric formsuch as:

More specifically, compounds of formula I where R^(2a) is —OH or—OP(O)(OH)₂, and R^(2b) is —CH₃ can contain at least two chiral centerswhen the “Z” moiety is —CH—; compounds of formula I where R^(2a) andR^(2b) are both —CH₃ or where R^(2a) and R^(2b) are taken together toform —CH₂—CH₂— can contain at least one chiral center when the “Z”moiety is —CH—; and compounds of formula I where R^(2a) is —OH or—OP(O)(OH)₂, and R^(2b) is —CH₃ can contain at least one chiral centerwhen the “Z” moiety is —N—. These chiral centers are indicated by thesymbols * and ** in the following formulas Ia, Ib, Ic, and Id(illustrated with R^(2a) as —OH):

In one stereoisomer of the compound of formula Ia, both carbon atomsidentified by the * and ** symbols have the (R) configuration. Thisembodiment of the invention is shown in formula Ia-1:

In this embodiment, compounds have the (R,R) configuration at the * and** carbon atoms or are enriched in a stereoisomeric form having the(R,R) configuration at these carbon atoms. In another stereoisomer ofthe compound of formula Ia, both carbon atoms identified by the * and **symbols have the (S) configuration. This embodiment of the invention isshown in formula Ia-2:

In this embodiment, compounds have the (S,S) configuration at the * and** carbon atoms or are enriched in a stereoisomeric form having the(S,S) configuration at these carbon atoms. In yet another stereoisomerof the compound of formula Ia, the carbon atom identified by thesymbol * has the (S) configuration and the carbon atom identified by thesymbol ** has the (R) configuration. This embodiment of the invention isshown in formula Ia-3:

In this embodiment, compounds have the (S,R) configuration at the * and** carbon atoms or are enriched in a stereoisomeric form having the(S,R) configuration at these carbon atoms. In still another stereoisomerof the compound of formula Ia, the carbon atom identified by thesymbol * has the (R) configuration and the carbon atom identified by thesymbol ** has the (S) configuration. This embodiment of the invention isshown in formula Ia-4:

In this embodiment, compounds have the (R,S) configuration at the * and** carbon atoms or are enriched in a stereoisomeric form having the(R,S) configuration at these carbon atoms.

In one stereoisomer of the compounds of formula Ib and Ic, the carbonatom identified by the ** symbol has the (R) configuration. Theseembodiments of the invention are shown in formulas Ib-1 and Ic-1,respectively:

In these embodiments, compounds have the (R) configuration at the **carbon atom or are enriched in a stereoisomeric form having the (R)configuration at this carbon atom. In another stereoisomer of thecompounds of formula Ib and Ic, the carbon atom identified by the **symbol has the (S) configuration. These embodiments of the invention areshown in formulas Ib-2 and Ic-2, respectively:

In these embodiments, compounds have the (S) configuration at the **carbon atom or are enriched in a stereoisomeric form having the (S)configuration at this carbon atom.

In one stereoisomer of the compound of formula Id, the carbon atomidentified by the * symbol has the (R) configuration. This embodiment ofthe invention is shown in formula Id-1:

In this embodiment, compounds have the (R) configuration at the * carbonatom or are enriched in a stereoisomeric form having the (R)configuration at this carbon atom. In another stereoisomer of thecompound of formula Id, the carbon atom identified by the * symbol hasthe (S) configuration. This embodiment of the invention is shown informula Id-2:

In this embodiment, compounds have the (S) configuration at the * carbonatom or are enriched in a stereoisomeric form having the (S)configuration at this carbon atom.

In some embodiments, in order to optimize the therapeutic activity ofthe compounds of the invention, e.g., to treat hypertension, it may bedesirable that the carbon atoms identified by the * and ** symbols havea particular configuration or are enriched in a stereoisomeric formhaving such configuration. Thus, in certain aspects, this inventionrelates to each individual enantiomer or to an enantiomer-enrichedmixture of enantiomers comprising predominately one enantiomer or theother enantiomer. In other embodiments, the compounds of the inventionare present as racemic mixtures of enantiomers.

The compounds of the invention, as well as those compounds used in theirsynthesis, may also include isotopically-labeled compounds, that is,where one or more atoms have been enriched with atoms having an atomicmass different from the atomic mass predominately found in nature.Examples of isotopes that may be incorporated into the compounds offormula I, for example, include, but are not limited to, ²H, ³H, ¹³C,¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ³⁶Cl, and ¹⁸F. Of particular interest arecompounds of formula I enriched in tritium or carbon-14 which can beused, for example, in tissue distribution studies; compounds of formulaI enriched in deuterium especially at a site of metabolism resulting,for example, in compounds having greater metabolic stability; andcompounds of formula I enriched in a positron emitting isotope, such as¹¹C, ¹⁸F, ¹⁵O and ¹³N, which can be used, for example, in PositronEmission Topography (PET) studies.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially available AutoNom software (MDL, San Leandro,Calif.).

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of theinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of the invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from the invention unless specifically indicated.

In one aspect, this invention relates to compounds of formula I:

R¹ is selected from —OR⁷ and —NR⁸R⁹. The R⁷ moiety is selected from:

H;

—C₁₋₈alkyl, e.g., —CH₃, —CH₂CH₃, —(CH₂)₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂,—(CH₂)₃CH₃, —(CH₂)₄CH₃, —(CH₂)₂CH(CH₃)₂, —(CH₂)₅CH₃, and —(CH₂)₆CH₃;

—C₁₋₃alkylene-C₆₋₁₀aryl, e.g., benzyl;

—C₁₋₃alkylene-C₁₋₉heteroaryl, e.g., —CH₂-pyridinyl and—(CH₂)₂-pyridinyl;

—C₃₋₇cycloalkyl, e.g., cyclopentyl;

—[(CH₂)₂O]₁₋₃CH₃, e.g., —(CH₂)₂OCH₃ and —[(CH₂)₂O]₂CH₃;

—C₁₋₆alkylene-OC(O)R¹⁰, e.g., —CH₂OC(O)CH₃, —CH₂OC(O)CH₂CH₃,—CH₂OC(O)(CH₂)₂CH₃, —CH₂CH(CH₃)OC(O)CH₂CH₃, —CH₂OC(O)OCH₃,—CH₂OC(O)OCH₂CH₃, —CH(CH₃)OC(O)OCH₂CH₃, —CH(CH₃)OC(O)O—CH(CH₃)₂,—CH₂CH(CH₃)OC(O)-cyclopentyl, —CH₂OC(O)O-cyclopropyl, —CH(CH₃)—OC(O)—O—cyclohexyl, —CH₂OC(O)O-cyclopentyl, —CH₂CH(CH₃)OC(O)-phenyl, —CH₂OC(O)O—phenyl, —CH₂OC(O)—CH[CH(CH₃)₂]—NH₂, —CH₂OC(O)—CH[CH(CH₃)₂]—NHC(O)OCH₃,and —CH(CH₃)OC(O)—CH(NH₂)CH₂COOCH₃;

—C₁₋₆alkylene-NR¹²R¹³, e.g., —(CH₂)₂—N(CH₃)₂,

—C₁₋₆alkylene-C(O)R³¹, e.g., —CH₂C(O)OCH₃, —CH₂C(O)O-benzyl, —CH₂C(O)—N(CH₃)₂, and

—C₀₋₆alkylenemorpholinyl, e.g., —(CH₂)₂-morpholinyl and—(CH₂)₃-morpholinyl:

—C₁₋₆alkylene-SO₂—C₁₋₆alkyl, e.g., —(CH₂)₂SO₂CH₃;

for example,

The R¹⁰ moiety is selected from:

—C₁₋₆alkyl, e.g., CH₃ and —CH₂CH₃;

—O—C₁₋₆alkyl, e.g., —OCH₃, O—CH₂CH₃, and O—CH(CH₃)₂;

—C₃₋₇cycloalkyl, e.g., cyclopentyl);

—O—C₃₋₇cycloalkyl, e.g., —O-cyclopropyl, —O-cyclohexyl, and—O-cyclopentyl; phenyl;

—O-phenyl;

—NR¹²R¹³;

—CH[CH(CH₃)₂]—NH₂;

—CH[CH(CH₃)₂]—NHC(O)O—C₁₋₆alkyl, e.g., —CH[CH(CH₃)₂]—NHC(O)OCH₃; and

—CH(NH₂)CH₂COOCH₃.

The R¹² and R¹³ moieties are independently selected from H, —C₁₋₆alkyl(e.g., CH₃), and benzyl. Alternately, the R¹² and R¹³ moieties can betaken together as —(CH₂)₃₋₆—, —C(O)— (CH₂)₃—, or —(CH₂)₂O(CH₂)₂—, forexample to form a group such as:

The R³¹ moiety is selected from —O—C₁₋₆alkyl, e.g., —OCH₃, —O-benzyl,and —NR¹²R¹³, e.g., —N(CH₃)₂, and

The R³² moiety is —C₁₋₆alkyl (e.g., —CH₃ and —C(CH₃)₃) or—C₀₋₆alkylene-C₆₋₁₀aryl.

The R⁸ moiety is selected from:

H;

—OH;

—OC(O)R¹⁴, e.g., —OC(O)CH₃, —OC(O)-phenyl, —OC(O)—OCH₂-phenyl,—OC(O)—CH₂O-phenyl, —OC(O)(NH₂), and —OC(O)[N(CH₃)₂;

—CH₂COOH;

—O-benzyl;

pyridyl; and

—OC(S)NR¹⁵R¹⁶, e.g., —OC(S)NH₂ and —OC(S)N(CH₃)₂.

The R¹⁴ moiety is selected from:

H;

—C₁₋₆alkyl, e.g., —CH₃;

—C₆₋₁₀aryl, e.g., phenyl;

—OCH₂—C₆₋₁₀aryl, e.g., —OCH₂-phenyl;

—CH₂O—C₆₋₁₀aryl, e.g., —CH₂O-phenyl; and

NR¹⁵R¹⁶, e.g., —NH₂ and N(CH₃)₂.

The R¹⁵ and R¹⁶ moieties are independently selected from H and—C₁₋₄alkyl.

The R⁹ is moiety selected from H, —C₁₋₆alkyl (e.g., —CH₃), and —C(O)R¹⁷(e.g., —C(O)H). The R¹⁷ moiety is selected from H, —C₁₋₆alkyl (e.g.,—CH₂CH₃), —C₃₋₇cycloalkyl (e.g., cyclopropyl), —C₆₋₁₀aryl (e.g.,phenyl), and —C₁₋₉heteroaryl (e.g., pyridine).

In addition, each alkyl group in R¹ is optionally substituted with 1 to8 fluoro atoms. For example, when R¹ is —OR⁷ and R⁷ is —C₁₋₈alkyl, R¹can also be a group such as —OCH(CH₃)CF₃, —OCH₂CF₂CF₃, —OCH(CF₃)₂,—O(CH₂)₂CF₃, —OCH(CH₂F)₂, —OC(CF₃)₂CH₃, and —OCH(CH₃)CF₂CF₃.

In one embodiment, R¹ is —OR⁷, and R⁷ is H or —C₁₋₈alkyl. In otherembodiments these compounds have formulas III-VIII.

In one embodiment, R¹ is selected from —OR⁷ and —NR⁸R⁹, R⁷ is H, R⁸ is Hor —OH, and R⁹ is H. I In other embodiments these compounds haveformulas III-VIII.

In another embodiment, R¹ is —OR⁷, where R⁷ is selected from —C₁₋₈alkyl,—C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl,—[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹⁰, —C₁₋₆alkylene-NR¹²R¹³,—C₁₋₆alkylene-C(O)R³¹, —C₀₋₆alkylenemorpholinyl;—C₁₋₆alkylene-SO₂—C₁₋₆alkyl;

In yet another embodiment, R¹ is —NR⁸R⁹; where R⁸ is selected from—OC(O)R¹⁴, —CH₂COOH, —O-benzyl, pyridyl, and —OC(S)NR¹⁵R¹⁶; and R⁹ is H.In yet another embodiment, R¹ is —NR⁸R⁹, where R⁸ is H or —OH; and R⁹ is—C₁₋₆alkyl or —C(O)R¹⁷. In yet another embodiment, R¹ is —NR⁸R⁹, whereR⁸ is selected from —OC(O)R¹⁴, —CH₂COOH, —O-benzyl, pyridyl, and—OC(S)NR¹⁵R¹⁶; and R⁹ is —C₁₋₆alkyl or —C(O)R¹⁷. In other embodimentsthese compounds have formulas III-VIII. In one aspect of the invention,these compounds may find particular utility as prodrugs or asintermediates in the synthetic procedures described herein. For example,in one embodiment, R¹ is —OR⁷ and R⁷ is —C₁₋₆alkylene-OC(O)R¹⁶, such asO—CH(CH₃)OC(O)—O-cyclohexyl:

making the compound a cilexetil ester; or R¹ is —OR⁷ and R⁷ is—C₀₋₆alkylenemorpholinyl such as —O—(CH₂)₂-morpholinyl:

making the compound a 2-morpholinoethyl or mofetil ester; or R¹ is —OR⁷and R⁷ is

such as —O—CH₂-5-methyl-[1,3]dioxol-2-one:

making the compound a medoxomil ester.

In one embodiment, R^(2a) is —OH and R^(2b) is —CH₃. In still anotherembodiment, R^(2a) is —OP(O)(OH)₂ and R^(2b) is —CH₃. In yet anotherembodiment, R^(2a) is —OC(O)CH(R³⁷)NH₂, where R³⁷ is selected from H,—CH(CH₃)₂, phenyl, and benzyl, and R^(2b) is —CH₃. In anotherembodiment, R^(2a) and R^(2b) are both —CH₃. In still anotherembodiment, R^(2a) and R^(2b) are taken together to form —CH₂—CH₂—.These can be depicted as formulas III, IV, V, VI, and VII respectively:

In other embodiment, R^(2a) is taken together with R⁷ to form —OCR¹⁸R¹⁹—or is taken together with R⁸ to form —OC(O)—. When R^(2a) is takentogether with R⁷ to form —OCR¹⁸R¹⁹—, this embodiment can be depicted as:

and when R¹⁸ and R¹⁹ are taken together to form ═O, this embodiment canbe depicted as:

When R^(2a) is taken together with R⁸ to form —OC(O)—, this embodimentcan be depicted as:

In one aspect of the invention, these compounds may find particularutility as prodrugs or as intermediates in the synthetic proceduresdescribed herein. Compounds where R^(2a) is —OP(O)(OH)₂ may also findutility as prodrugs.

The “Z” moiety is —CH— or —N—, which can be depicted as formulas Ie andIf, respectively:

In one embodiment Z is —CH—, R^(2a) is —OH, and R^(2b) is —CH₃. In oneembodiment Z is —CH—, R^(2a) is —OP(O)(OH)₂, and R^(2b) is —CH₃. In oneembodiment Z is —CH—, R^(2a) is —OC(O)CH(R³⁷)NH₂, and R^(2b) is —CH₃. Inanother embodiment Z is —CH— and R^(2a) and R^(2b) are both —CH₃. In yetanother embodiment Z is —CH— and R^(2a) and R^(2b) are taken together toform —CH₂—CH₂—. These embodiments can be depicted as formulas Ma, IVa,Va, VIa, and VIIa, respectively:

In another embodiment Z is —N—, R^(2a) is —OH, and R^(2b) is —CH₃. Inone embodiment Z is —N—, R^(2a) is —OP(O)(OH)₂, and R^(2b) is —CH₃. Inone embodiment Z is —N—, R^(2a) is —OC(O)CH(R³⁷)NH₂, and R^(2b) is —CH₃.In another embodiment Z is —N— and R^(2a) and R^(2b) are both —CH₃. Inyet another embodiment Z is —N— and R^(2a) and R^(2b) are taken togetherto form —CH₂—CH₂—. These embodiments can be depicted as formulas IIIb,IVb, Vb, VIb, and VIIb, respectively:

The “X” moiety is a —C₁₋₉heteroaryl, and the point of attachment is atany available carbon or nitrogen ring atom. Note that in someembodiments, R³ and/or R⁴ may be absent. When present, R³ is on anyavailable carbon atom. When present, R⁴ is on any available carbon atomor nitrogen atom. Exemplary —C₁₋₉heteroaryl rings include, by way ofillustration and not limitation:

pyrazole rings such as:

specific examples of which include:

imidazole rings such as:

specific examples of which include:

triazole rings, including 1,2,3-triazoles such as:

as well as 1,2,4-triazoles such as:

benzotriazole rings such as:

specific examples of which include:

furan rings:

specific examples of which include:

pyrrole rings:

specific examples of which include:

tetrazole rings such as:

pyrazine rings:

a specific example of which includes:

thiophene rings:

specific examples of which include:

oxazole rings:

specific examples of which include:

isoxazole rings:

specific examples of which include:

thiazole rings:

specific examples of which include:

isothiazole rings:

oxadiazole rings, including [1,2,4]oxadiazoles such as:

as well as [1,2,3]oxadiazoles such as:

and [1,3,4]oxadiazoles:

thiadiazole rings, including [1,2,4]thiadiazoles such as:

as well as [1,2,3]thiadiazoles such as:

and [1,3,4]thiadiazoles:

pyridazine rings:

pyridine rings:

specific examples of which include:

pyrimidine rings:

specific examples of which include:

pyran rings such as:

benzimidazole rings such as:

specific examples of which include:

benzoxazole rings such as:

specific examples of which include:

benzothiazole rings such as:

specific examples of which include:

pyridylimidazole rings such as:

a specific example of which includes:

and pyridyltriazole rings such as:

specific examples of which include:

In one particular embodiment, X is selected from pyrazole, imidazole,triazole, benzotriazole, furan, pyrrole, tetrazole, pyrazine, thiophene,oxazole, isoxazole, thiazole, isothiazole, oxadiazole, thiadiazole,pyridazine, pyridine, pyrimidine, pyran, benzimidazole, benzoxazole,benzothiazole, pyridylimidazole, and pyridyltriazole. In otherembodiments these compounds have formulas III-VI.

It is understood that some —C₁₋₉heteroaryl rings can exist in atautomeric form, and that such tautomeric forms are part of theinvention and are encompassed by the term “heteroaryl.” Therefore, if acompound is depicted with a —C₁₋₉heteroaryl ring, it is understood thatthe compound can also exist in a tautomeric form and vice versa, andthat both forms are covered by the invention.

—C₁₋₉heteroaryl ring exemplary ring exemplary tautomer(s) pyrazole

imidazole

triazole

oxazole

thiazole

isothiazole

oxadiazole

thiadiazole

pyridazine

In one particular embodiment, X is selected from pyrazole, triazole,benzotriazole, tetrazole, oxazole, isoxazole, pyrimidine, andpyridyltriazole. In other embodiments these compounds have formulasIII-VII. In still another embodiment X is triazole, and in one specificembodiment, have formula VIII:

where Z, R¹, R^(2a), R^(2b), R³—R⁶, a, and b are as defined for formulaI.

The R³ moiety can be absent. When present, R³ is attached to a carbonatom in the “X” group, and is selected from:

H;

halo, e.g., chloro and fluoro;

—C₀₋₅alkylene-OH, e.g., —OH, —CH₂OH, —CH(OH)CH₃, and —C(CH₃)₂—OH;

—NH₂;

—C₁₋₆alkyl, e.g., —CH₃, —(CH₂)₂CH₃, —CH(CH₃)₂, and —(CH₂)₃—CH₃;

—CF₃;

—C₃₋₇cycloalkyl, e.g., cyclopropyl and cyclohexyl;

—C₀₋₂alkylene-O—C₁₋₆alkyl, e.g., —OCH₃, —OCH₂CH₃, —CH₂—OCH₃, and—(CH₂)₂—OCH₃;

—C(O)R²⁰, e.g., —C(O)H and —C(O)CH₃;

—C₀₋₁alkylene-COOR²¹, e.g., —COOH, —CH₂—COOH, —C(O)O—CH₂CH₃, —C(O)O—(CH₂)₂OCH₃—C(O)O—CH₂OC(O)CH₃, —CH₂—C(O)O—CH₂OC(O)CH₃,—C(O)O—CH₂OC(O)O—CH₃, —CH₂—C(O)O—CH₂OC(O)O—CH₃,—C(O)O—CH(CH₃)OC(O)O—CH₂CH₃, —C(O)O—CH(CH₃)OC(O)O—CH(CH₃)₂,—C(O)O—CH₂CH(CH₃)OC(O)-cyclopentyl, —C(O)O— CH₂OC(O)O-cyclopropyl,—C(O)O—CH(CH₃)—OC(O)—O-cyclohexyl, —C(O)O—CH₂OC(O)O— cyclopentyl,—C(O)O—CH₂CH(CH₃)OC(O)-phenyl, —C(O)O—CH₂OC(O)O-phenyl,—C(O)O—CH₂-pyridine, —C(O)O—CH₂-pyrrolidine, —C(O)O—(CH₂)₂-morpholinyl,—C(O)O—(CH₂)₃-morpholinyl, and —C(O)O—(CH₂)₂—SO₂—CH₃;

—C(O)NR²²R²³, e.g., —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —C(O)NH—(CH₂)₂CH₃, —C(O)NH—CH₂COOH, —C(O)NH—(CH₂)₂—OH, —C(O)NH—(CH₂)₂—N(CH₃)₂,—C(O)NH-cyclopropyl, —C(O)NH—(CH₂)₂-imidazolyl, —C(O)N(CH₃)—CH₂CH(CH₃)₂,and —C(O)N(CH₃)[(CH₂)₂OCH₃];

—NHC(O)R²⁴, e.g., —NHC(O)—CH₂CH₃, —NHC(O)—(CH₂)₃CH₃, —NHC(O)O—CH₂CH₃,—NHC(O)—CH₂—OCH₃, —NHC(O)-2-methoxyphenyl, —NHC(O)-2-chlorophenyl, and—NHC(O)-2-pyridine;

═O;

—NO₂;

—C(CH₃)═N(OH);

phenyl optionally substituted with one or two groups independentlyselected from halo, —OH, —CF₃, —OCH₃, —NHC(O)CH₃, and phenyl (e.g.,phenyl, 2-chlorophenyl, 2-fluorophenyl, 2-hydroxyphenyl,2-trifluoromethylphenyl, 2-methoxyphenyl, 3-chlorophenyl,3-fluorophenyl, 3-methoxyphenyl, 3—NHC(O)CH₃-phenyl, 4-chlorophenyl,4-fluorophenyl, 4-methoxyphenyl, 4-biphenyl, 2,5-dichlorophenyl,2,5-dimethoxyphenyl, 2,4-dichlorophenyl, 2-methoxy, 5-fluorophenyl, and3,4-dichlorophenyl);

naphthalenyl;

pyridinyl;

pyrazinyl;

pyrazolyl optionally substituted with methyl;

thiophenyl optionally substituted with methyl or halo (e.g., chloro);

furanyl; and

—CH₂-morpholinyl.

The R²⁰ moiety is selected from H and —C₁₋₆alkyl (e.g., —CH₃). The R²¹moiety is selected from:

H;

—C₁₋₆alkyl, e.g., —CH₃ and —CH₂CH₃;

—C₁₋₃alkylene-C₆₋₁₀aryl;

—C₁₋₃alkylene-C₁₋₉heteroaryl, e.g., —CH₂-pyridine;

—C₃₋₇cycloalkyl;

—[(CH₂)₂O]₁₋₃CH₃, e.g., —(CH₂)₂OCH₃;

—C₁₋₆alkylene-OC(O)R²⁵, e.g., —CH₂OC(O)CH₃, —CH₂OC(O)O—CH₃,—CH₂OC(O)O—CH₃, —CH(CH₃)OC(O)O—CH₂CH₃, —CH(CH₃)OC(O)O—CH(CH₃)₂,—CH₂CH(CH₃)OC(O)— cyclopentyl, —CH₂OC(O)O-cyclopropyl,—CH(CH₃)—OC(O)—O-cyclohexyl, —CH₂OC(O)O— cyclopentyl,—CH₂CH(CH₃)OC(O)-phenyl, and —CH₂OC(O)O-phenyl;

—C₁₋₆ alkylene-NR²²R²⁸, e.g., —CH₂-pyrrolidine;

—C₁₋₆alkylene-C(O)R³³;

—C₀₋₆alkylenemorpholinyl, e.g., —(CH₂)₂-morpholinyl and—(CH₂)₃-morpholinyl:

—C₁₋₆alkylene-SO₂—C₁₋₆alkyl, e.g., —(CH₂)₂—SO₂—CH₃;

e.g.,

The R²² and R²³ moieties are independently selected from:

H;

—C₁₋₆alkyl, e.g., —CH₃ and —(CH₂)₂CH₃;

—CH₂COOH;

—(CH₂)₂OH;

—(CH₂)₂OCH₃;

—(CH₂)₂SO₂NH₂;

—(CH₂)₂N(CH₃)₂;

—C₀₋₁alkylene-C₃₋₇cycloalkyl, e.g., cyclopropyl and —CH₂-cyclopropyl;and

—(CH₂)₂-imidazolyl.

R²² and R²³ may also be taken together to form a saturated or partiallyunsaturated —C₃₋₅heterocycle optionally substituted with halo, —OH,—COOH, or —CONH₂, and optionally containing an oxygen atom in the ring.Saturated —C₃₋₅heterocycles include azetidine, pyrrolidine, piperidineand morpholine, such that exemplary R³ groups include:

Partially unsaturated —C₃₋₅heterocycles include 2,5-dihydro-1H-pyrrole,such that exemplary R³ groups include:

The R²⁴ moiety is selected from:

—C₁₋₆alkyl, e.g., —CH₂CH₃ and —(CH₂)₃CH₃;

—C₀₋₁alkylene-O—C₁₋₆alkyl, e.g., —O—CH₂CH₃ and —CH₂—OCH₃;

phenyl optionally substituted with halo or —OCH₃, e.g., -2chlorophenylor -2-methoxyphenyl; and

—C₁₋₉heteroaryl, e.g., 2-pyridine.

R²⁵ is selected from:

—C₁₋₆alkyl, e.g., —CH₃, —CH₂CH₃, and —(CH₂)₃CH₃;

—O—C₁₋₆alkyl, e.g., —OCH₃, —OCH₂CH₃, and —OCH(CH₃)₂;

—C₃₋₇cycloalkyl, e.g., cyclopentyl;

—O—C₃₋₇cycloalkyl, e.g., —O-cyclopropyl, —O-cyclopentyl, and—O-cyclohexyl; phenyl;

—O-phenyl;

NR²⁷R²⁸;

—CH[CH(CH₃)₂]—NH₂;

—CH[CH(CH₃)₂]—NHC(O)O—C₁₋₆alkyl, e.g., —CH[CH(CH₃)₂]—NHC(O)OCH₃; and

—CH(NH₂)CH₂COOCH₃.

R²⁷ and R²⁸ are independently selected from H, —C₁₋₆alkyl, and benzyl,or R²⁷ and R²⁸ are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or—(CH₂)₂O(CH₂)₂—; R³³ is selected from —O—C₁₋₆alkyl, —O-benzyl, and—NR²⁷R²⁸; and R³⁴ is —C₁₋₆alkyl (e.g., —CH₃ and —C(CH₃)₃) or—C₀₋₆alkylene-C₆₋₁₀aryl.

In addition, each alkyl group in R³ is optionally substituted with 1 to8 fluoro atoms. For example, when R³ is —C₀₋₁alkylene-COOR²¹ and R²¹ is—C₁₋₆alkyl, R³ can also be a group such as —COOCH(CH₃)CF₃,—COOCH₂CF₂CF₃, —COOCH(CF₃)₂, —COO(CH₂)₂CF₃, —COOCH(CH₂F)₂,—COOC(CF₃)₂CH₃, and —COOCH(CH₃)CF₂CF₃.

In one embodiment, R³ is absent or is selected from H, halo,—C₀₋₅alkylene-OH, —C₀₋₁alkylene-O—C₁₋₆alkyl, —C(O)R²⁰,C₀₋₁alkylene-COOR²¹, —C(O)NR²²R²³, ═O, and pyrazinyl; R²⁰ is —C₁₋₆alkyl;R²¹ is selected from H and —C₁₋₆alkyl; R²² and R²³ are independentlyselected from H, —C₁₋₆alkyl, —(CH₂)₂OCH₃, and —(CH₂)₂-imidazolyl; or R²²and R²³ are taken together to form a saturated —C₃₋₅heterocycleoptionally substituted with —OH or —COOH, and optionally containing anoxygen atom in the ring. In other embodiments these compounds haveformulas III-VIII.

In one embodiment, R³ is absent or is selected from H; halo;—C₀₋₅alkylene-OH; —NH₂; —C₁₋₆alkyl; —CF₃; —C₃₋₂cycloalkyl;—C₀₋₂alkylene-O—C₁₋₆alkyl; —C(O)R²⁰; —C₀₋₁alkylene-COOR²¹; —C(O)NR²²R²³; —NHC(O)R²⁴; ═O; —NO₂; —C(CH₃)═N(OH); phenyl optionallysubstituted with one or two groups independently selected from halo,—OH, —CF₃, —OCH₃, —NHC(O)CH₃, and phenyl; naphthalenyl; pyridinyl;pyrazinyl; pyrazolyl optionally substituted with methyl; thiophenyloptionally substituted with methyl or halo; furanyl; and—CH₂-morpholinyl; and R²¹ is H. In other embodiments these compoundshave formulas III-VIII.

In another embodiment, R³ is —C₀₋₁alkylene-COOR²¹, and R²¹ is selectedfrom —C₁₋₆ alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl,—C₃₋₂cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R²⁵;—C₁₋₆alkylene-NR²⁷R²⁸, C₁₋₆alkylene-C(O)R³³, —C₀₋₆alkylenemorpholinyl,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

In one aspect of the invention, these compounds may find particularutility as prodrugs or as intermediates in the synthetic proceduresdescribed herein. In other embodiments these compounds have formulasIII-VIII.

The R⁴ moiety can be absent. When present, R⁴ is attached to a carbon ornitrogen atom in the “X” group, and is selected from:

H;

—OH;

—C₁₋₆alkyl, e.g., —CH₃;

—C₁₋₂alkylene-COOR³⁵, e.g., —CH₂COOH and —(CH₂)₂—COOH;

—CH₂OC(O)CH(R³⁶)NH₂, e.g., —CH₂OC(O)CH[CH(CH₃)₂]NH₂;

—OCH₂OC(O)CH(R³⁶)NH₂, e.g., —OCH₂OC(O)CH[CH(CH₃)₂]NH₂;

—OCH₂OC(O)CH₃;

—CH₂OP(O)(OH)₂;

—CH₂CH(OH)CH₂OH;

—CH[CH(CH₃)₂]—NHC(O)O—C₁₋₆alkyl;

pyridinyl; and

phenyl or benzyl optionally substituted with one or more groups selectedfrom halo, —COOR³⁵, —OCH₃, —OCF₃, and —SCF₃ (e.g., 4-chlorophenyl,3-methoxyphenyl, 2,4-dichlorophenyl, 3,4-dichlorophenyl, 2-chloro,5-fluorophenyl, 3-trifluoromethoxy, 4-chlorophenyl,3-trifluoromethylsulfanyl, 4-chlorophenyl, 2,6-difluoro, 4-chlorophenyl,2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 3-carboxybenzyl,4-carboxybenzyl, 3-methoxybenzyl, 2-chloro, 5-fluorobenzyl, 3-chloro,5-fluorobenzyl, 2-fluoro, 4-chlorobenzyl, 3-chloro, 4-fluorobenzyl,3—OCF3,4-chlorobenzyl, 3-SCF3,4-chlorobenzyl, 2,6-difluoro,3-chlorobenzyl, 2,6-difluoro, 4-chlorobenzyl, and 2,3,5,6-tetrafluoro,4-methoxy benzyl).

The R³⁵ moiety is selected from:

H;

—C₁₋₆alkyl, e.g., —CH₃ and —CH₂CH₃;

—C₁₋₃alkylene-C₆₋₁₀aryl;

—C₁₋₃alkylene-C₁₋₉heteroaryl, e.g., —CH₂-pyridine;

—C₃₋₇cycloalkyl;

—[(CH₂)₂O]₁₋₃CH₃, e.g., —(CH₂)₂OCH₃;

—C₁₋₆alkylene-OC(O)R²⁵, e.g., —CH₂OC(O)CH₃, —CH₂OC(O)O—CH₃,—CH₂OC(O)O—CH₃, —CH(CH₃)OC(O)O—CH₂CH₃, —CH(CH₃)OC(O)O—CH(CH₃)₂,—CH₂CH(CH₃)OC(O)-cyclopentyl, —CH₂OC(O)O-cyclopropyl,—CH(CH₃)—OC(O)—O-cyclohexyl, —CH₂OC(O)O— cyclopentyl,—CH₂CH(CH₃)OC(O)-phenyl, and —CH₂OC(O)O-phenyl;

—C₁₋₆alkylene-NR²²R²⁸, e.g., —CH₂-pyrrolidine;

—C₁₋₆alkylene-C(O)R³³;

—C₀₋₆alkylenemorpholinyl, e.g., —(CH₂)₂-morpholinyl and—(CH₂)₃-morpholinyl:

—C₁₋₆alkylene-SO₂—C₁₋₆alkyl, e.g., —(CH₂)₂—SO₂—CH₃;

e.g.,

The R²⁵, R²⁷, R²⁸, R³³, and R³⁴ moieties are defined above. The R³⁶moiety is selected from H, —CH(CH₃)₂, phenyl, and benzyl.

In addition, each alkyl group in R⁴ is optionally substituted with 1 to8 fluoro atoms. For example, when R⁴ is —C₁₋₂alkylene-COOR³⁵ and R³⁵ is—C₁₋₆alkyl, R⁴ can also be a group such as —COOCH(CH₃)CF₃,—COOCH₂CF₂CF₃, —COOCH(CF₃)₂, —COO(CH₂)₂CF₃, —COOCH(CH₂F)₂,—COOC(CF₃)₂CH₃, and —COOCH(CH₃)CF₂CF₃.

The R⁴ moiety can also be taken together with R³ to form-phenylene-O—(CH₂)₁₋₃— or -phenylene-O—CH₂—CHOH—CH₂—. For purposes ofillustration only, these embodiments are depicted below with X beingpyrazole. It is understood that other X groups can be used also.

In another particular embodiment, R⁴ is selected from H and —OH. Inother embodiments these compounds have formulas III-VIII.

In one embodiment, R⁴ is absent or is selected from H; —OH; —C₁₋₆alkyl;—C₁₋₂alkylene-COOR³⁵; —CH₂OC(O)CH(R³⁶)NH₂, —CH₂CH(OH)CH₂OH; pyridinyl;and phenyl or benzyl optionally substituted with one or more groupsselected from halo, —COOR³⁵, —OCH₃, —OCF₃, and —SCF₃; and R³⁵ is H. Inother embodiments these compounds have formulas III-VIII.

In another embodiment, R⁴ is selected from —OCH₂OC(O)CH₃;—CH₂OP(O)(OH)₂; —C₁₋₂alkylene-COOR³⁵; and phenyl or benzyl substitutedwith at least one —COOR³⁵ group; where R³⁵ is selected from —C₁₋₆alkyl,—C₁₋₃ alkylene-C₆₋₁₀aryl, —C₁₋₃ alkylene-C₁₋₉heteroaryl,—C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R²⁵;—C₁₋₆alkylene-NR^(27R28,)—C₁₋₆alkylene-C(O)R³³,—C₀₋₆alkylenemorpholinyl, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

In one aspect of the invention, these compounds may find particularutility as prodrugs or as intermediates in the synthetic proceduresdescribed herein. In other embodiments these compounds have formulasIII-VIII.

The numbering for the R⁵ and R⁶ groups is as follows:

The integer “a” is 0 or 1. The R⁵ moiety, when present, is selected fromhalo, —CH₃, —CF₃, and —CN. In one embodiment, a is 0. In anotherembodiment, a is 1, and R⁵ is halo, such as 3-chloro or 3-fluoro. Theinteger “b” is 0 or an integer from 1 to 3. The R⁶ moiety, when present,is independently selected from halo, —OH, —CH₃, —OCH₃, and —CF₃. In oneembodiment, b is 0. In another embodiment, b is 1 and R⁶ is selectedfrom halo, —OH, —CH₃, —OCH₃, and —CF₃, such 2′-chloro, 3′-chloro,2′-fluoro, 3′-fluoro, 2′-hydroxy, 3′-hydroxy, 3′-methyl, 2′-methoxy, or3′-trifluoromethyl. In another embodiment, b is 1 and R⁶ is selectedfrom halo, —OH, and —OCH₃, such 2′-chloro, 3′-chloro, 2′-fluoro,3′-fluoro, 2′-hydroxy, or 2′-methoxy. In one embodiment, b is 2 and R⁶is 2′-fluoro-5′-chloro, 2′,5′-dichloro, 2′,5′-difluoro,2′-methyl-5′-chloro, 3′-fluoro-5′-chloro, 3′-hydroxy-5′-chloro,3′,5′-dichloro, 3′,5′-difluoro, 2′-methoxy-5′-chloro,2′-methoxy-5′-fluoro, 2′-hydroxy-5′-fluoro, 2′-fluoro-3′-chloro,2′-hydroxy-5′-chloro, or 2′-hydroxy-3′-chloro; and in anotherembodiment, b is 2 and each R⁶ is independently selected from halo and—CH₃, for example, 2′-fluoro-5′-chloro, 2′,5′-difluoro, 2′,5′-dichloro,3′,5′-dichloro, and 2′-methyl-5′-chloro. In another embodiment, b is 3and each R⁶ is independently halo or —CH₃, such as2′-methyl-3′,5′-dichloro or 2′-fluoro-3′-methyl-5′-chloro. In yetanother embodiment, a is 1 and b is 1 and R⁵ and R⁶ are independentlyhalo, for example, 3-chloro and 3′chloro. In other embodiments thesecompounds have formulas III-VIII. Of particular interest are compoundsof the formulas:

The methylene linker on the biphenyl is optionally substituted with oneor two —C₁₋₆alkyl groups or cyclopropyl. For example, in one embodiment,the methylene linker on the biphenyl is unsubstituted; in anotherembodiment, the methylene linker on the biphenyl is substituted with one—C₁₋₆alkyl group (e.g., —CH₃); and in yet another embodiment, themethylene linker on the biphenyl is substituted with two —C₁₋₆alkylgroups (e.g., two —CH₃ groups); in another embodiment, the methylenelinker on the biphenyl is substituted with a cyclopropyl group. Theseembodiments are depicted, respectively, as:

In another embodiment, R¹ is —OR⁷; R⁷ is H or —C₁₋₆alkyl; X is selectedfrom pyrazole, triazole, benzotriazole, tetrazole, oxazole, isoxazole,pyrimidine, and pyridyltriazole; R³ is absent or is selected from H,halo, —C₀₋₅alkylene-OH, —C₀₋₁alkylene-O—C₁₋₆alkyl, —C(O)R²⁰,C₀₋₁alkylene-COOR²¹, —C(O)NR²²R²³, ═O, and, pyrazinyl; R²⁰ is—C₁₋₆alkyl; R²¹ is H or —C₁₋₆alkyl; R²² and R²³ are independentlyselected from H, —C₁₋₆alkyl, —(CH₂)₂OCH₃, and —(CH₂)₂-imidazolyl; or R²²and R²³ are taken together to form a saturated —C₃₋₅heterocycleoptionally substituted with —OH or —COOH, and optionally containing anoxygen atom in the ring; R⁴ is selected from H and —OH; a is 0; b is 0;or b is 1 and R⁶ is selected from halo, —OH, and —OCH₃; or b is 2 andeach R⁶ is independently selected from halo and —CH₃; and Z, R^(ea), andR^(2b) are as defined for formula I.

In still another embodiment, R¹ is —OR⁷; R^(2a) is; —OH and R^(2b) is—CH₃; or R^(2a) and R^(2b) are both —CH₃; or R^(2a) and R^(2b) are takentogether to form —CH₂—CH₂—; X is selected from pyrazole, triazole,isoxazole, and pyrimidine; R³ is selected from H, —C₀₋₅alkylene-OH, and—C₀₋₁alkylene-COOR²¹; R²¹ is H or —C₁₋₆alkyl; R⁴ is selected from H and—OH; a is 0; and b is 0; or b is 1 and R⁶ is halo; or b is 2 and each R⁶is independently selected from halo and —CH₃; and R⁷ is as defined forformula I.

A particular group of compounds of formula I are those disclosed in U.S.Provisional Application No. 61/443,828, filed on Feb. 17, 2011. Thisgroup includes compounds of formula II:

where: R¹ is selected from —OR⁷ and NR⁸R⁹; R⁷ is selected from H;—C₁₋₆alkyl; —C₁₋₃alkylene-C₆₋₁₀aryl; —C₁₋₃alkylene-C₁₋₉heteroaryl;—C₃₋₇cycloalkyl; —[(CH₂)₂O]₁₋₃CH₃; —C₁₋₆alkylene-OC(O)R¹⁰;—CH₂-pyridine; —CH₂-pyrrolidine; —C₀₋₆alkylenemorpholine;—C₁₋₆alkylene-SO₂—C₁₋₆alkyl;

where R¹⁰ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹²R¹³, and —CH(NH₂)CH₂COOCH₃;and R¹² and R¹³ are independently selected from H, —C₁₋₆alkyl, andbenzyl, or R¹² and R¹³ are taken together as —(CH₂)₃₋₆—; R⁸ is selectedfrom H; —OH; —OC(O)R¹⁴; —CH₂COOH; —O-benzyl; pyridyl; and —OC(S)NR¹⁵R¹⁶;where R¹⁴ is selected from H, —C₁₋₆alkyl, —C₆₋₁₀aryl, —OCH₂—C₆₋₁₀aryl,—CH₂O—C₆₋₁₀aryl, and NR¹⁵R¹⁶; and R¹⁵ and R¹⁶ are independently selectedfrom H and —C₁₋₄alkyl; R⁹ is selected from H; —C₁₋₆alkyl; and —C(O)R¹⁷;where R¹⁷ is selected from H; —C₁₋₆alkyl; —C₃₋₇cycloalkyl; —C₆₋₁₀aryl;and —C₁₋₉heteroaryl; R^(2a) is —OH and R^(2b) is —CH₃; or R^(2a) andR^(2b) are both —CH₃; or R^(2a) and R^(2b) are taken together to form—CH₂—CH₂—; Z is selected from —CH— and —N—; X is a —C₁₋₉heteroaryl or apartially unsaturated —C₃₋₅heterocycle; R³ is absent or is selected fromH; halo; —C₀₋₅alkylene-OH; —NH₂; —C₁₋₆alkyl; —CF₃; —C₃₋₇cycloalkyl;—C₀₋₁alkylene-O—C₁₋₆alkyl; —C(O)R²⁰; —C₀₋₁alkylene-C(O)OR²¹;—C(O)NR²²R²³; —NHC(O)R²⁴; phenyl optionally substituted with one groupselected from halo, —CF₃, —OCH₃, —NHC(O)CH₃, and phenyl; napthyl;pyridine; pyrazine; pyrazole optionally substituted with methyl;thiophene optionally substituted with methyl; and furan; and R³, whenpresent, is attached to a carbon atom; R²⁰ is selected from H and—C₁₋₆alkyl; R²¹ is selected from H; —C₁₋₆alkyl; —C₁₋₃alkylene-C₆₋₁₀aryl;—C₁₋₃alkylene-C₁₋₉heteroaryl; —C₃₋₇cycloalkyl; —[(CH₂)₂O]₁₋₃CH₃;—C₁₋₆alkylene-OC(O)R²⁵; —CH₂-pyridine; —CH₂-pyrrolidine;—C₀₋₆alkylenemorpholine; —C₁₋₆alkylene-SO₂—C₁₋₆alkyl;

where R²⁵ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR²⁷R²⁸, and —CH(NH₂)CH₂COOCH₃;and R²⁷ and R²⁸ are independently selected from H, —C₁₋₆alkyl, andbenzyl, or R²⁷ and R²⁸ are taken together as —(CH₂)₃₋₆—; R²² and R²³ areindependently selected from H; —C₁₋₆alkyl; —CH₂COOH; —(CH₂)₂OH;—(CH₂)₂OCH₃; —(CH₂)₂SO₂NH₂; —(CH₂)₂N(CH₃)₂; —C₃₋₇cycloalkyl; and—(CH₂)₂-imidazole; or R²² and R²³ are taken together to form a saturatedor partially unsaturated —C₃₋₅heterocycle optionally substituted with—OH, —COOH, or —CONH₂; and optionally containing an oxygen atom in thering; R²⁴ is selected from —C₁₋₆alkyl; —C₀₋₁alkylene-O—C₁₋₆alkyl; phenyloptionally substituted with —OCH₃; and —C₁₋₉heteroaryl; R⁴ is absent oris selected from H; —C₁₋₆alkyl; and phenyl or benzyl substituted withone or more groups selected from halo, —COOH, —OCH₃, —OCF₃, and —SCF₃;and R⁴, when present, is attached to a carbon or nitrogen atom; a is 0or 1; R⁵ is halo, —CH₃, or —CF₃; and b is 0 or 1; R⁶ is halo; or apharmaceutically acceptable salt thereof.

In addition, particular compounds of formula I that are of interestinclude those set forth in the Examples below, as well aspharmaceutically acceptable salts thereof.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods, the proceduresset forth in the Examples, or by using other methods, reagents, andstarting materials that are known to those of ordinary skill in the art.Although the following procedures may illustrate a particular embodimentof the invention, it is understood that other embodiments of theinvention can be similarly prepared using the same or similar methods orby using other methods, reagents and starting materials known to thoseof ordinary skill in the art. It will also be appreciated that wheretypical or preferred process conditions (for example, reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. In some instances, reactions were conducted at roomtemperature and no actual temperature measurement was taken. It isunderstood that room temperature can be taken to mean a temperaturewithin the range commonly associated with the ambient temperature in alaboratory environment, and will typically be in the range of about 18°C. to about 30° C. In other instances, reactions were conducted at roomtemperature and the temperature was actually measured and recorded.While optimum reaction conditions will typically vary depending onvarious reaction parameters such as the particular reactants, solventsand quantities used, those of ordinary skill in the art can readilydetermine suitable reaction conditions using routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well-known in the art.Protecting groups other than those illustrated in the proceduresdescribed herein may be used, if desired. For example, numerousprotecting groups, and their introduction and removal, are described inT. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis,Fourth Edition, Wiley, New York, 2006, and references cited therein.

Carboxy-protecting groups are suitable for preventing undesiredreactions at a carboxy group, and examples include, but are not limitedto, methyl, ethyl, t-butyl, benzyl (Bn), p-methoxybenzyl (PMB),9-fluorenylmethyl (Fm), trimethylsilyl (TMS), t-butyldimethylsilyl(TBDMS), diphenylmethyl (benzhydryl, DPM) and the like. Amino-protectinggroups are suitable for preventing undesired reactions at an aminogroup, and examples include, but are not limited to, t-butoxycarbonyl(BOC), trityl (Tr), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl(Fmoc), formyl, trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), andthe like. Hydroxyl-protecting groups are suitable for preventingundesired reactions at a hydroxyl group, and examples include, but arenot limited to C₁₋₆alkyls, say′ groups including triC₁₋₆alkylsilylgroups, such as trimethylsilyl (TMS), triethylsilyl (TES), andtert-butyldimethylsilyl (TBDMS); esters (acyl groups) includingC₁₋₆alkanoyl groups, such as formyl, acetyl, and pivaloyl, and aromaticacyl groups such as benzoyl; arylmethyl groups such as benzyl (Bn),p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl(benzhydryl, DPM); and the like.

Standard deprotection techniques and reagents are used to remove theprotecting groups, and may vary depending upon which group is used. Forexample, sodium or lithium hydroxide is commonly used when thecarboxy-protecting group is methyl, an acid such as TFA or HCl iscommonly used when the carboxy-protecting group is ethyl or t-butyl, andH₂/Pd/C may be used when the carboxy-protecting group is benzyl. A BOCamino-protecting group can be removed using an acidic reagent such asTFA in DCM or HCl in 1,4-dioxane, while a Cbz amino-protecting group canbe removed by employing catalytic hydrogenation conditions such as H₂ (1atm) and 10% Pd/C in an alcoholic solvent (“H₂/Pd/C”). H₂/Pd/C iscommonly used when the hydroxyl-protecting group is benzyl, while NaOHis commonly used when the hydroxyl-protecting group is an acyl group.

Suitable bases for use in these schemes include, by way of illustrationand not limitation, potassium carbonate, calcium carbonate, sodiumcarbonate, triethylamine, pyridine, 1,8-diazabicyclo-[5.4.0]undec-7-ene(DBU), N,N-diisopropylethylamine (DIPEA), 4-methylmorpholine, sodiumhydroxide, potassium hydroxide, potassium t-butoxide, and metalhydrides.

Suitable inert diluents or solvents for use in these schemes include, byway of illustration and not limitation, tetrahydrofuran (THF),acetonitrile (MeCN), N,N-dimethylformamide (DMF), N,N-dimethylacetamide(DMA), dimethyl sulfoxide (DMSO), toluene, dichloromethane (DCM),chloroform (CHCl₃), carbon tetrachloride (CCl₄), 1,4-dioxane, methanol,ethanol, water, and the like.

Suitable carboxylic acid/amine coupling reagents includebenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate(PyBOP), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU), 1,3-dicyclohexylcarbodiimide (DCC),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI),carbonyldiimidazole (CDI), 1-hydroxybenzotriazole (HOBt), and the like.Coupling reactions are conducted in an inert diluent in the presence ofa base such as DIPEA, and are performed under conventional amidebond-forming conditions.

All reactions are typically conducted at a temperature within the rangeof about −78 C to 100° C., for example at room temperature. Reactionsmay be monitored by use of thin layer chromatography (TLC), highperformance liquid chromatography (HPLC), and/or LCMS until completion.Reactions may be complete in minutes, or may take hours, typically from1-2 hours and up to 48 hours. Upon completion, the resulting mixture orreaction product may be further treated in order to obtain the desiredproduct. For example, the resulting mixture or reaction product may besubjected to one or more of the following procedures: concentrating orpartitioning (for example, between EtOAc and water or between 5% THF inEtOAc and 1M phosphoric acid); extraction (for example, with EtOAc,CHCl₃, DCM, chloroform); washing (for example, with saturated aqueousNaCl, saturated NaHCO₃, Na₂CO₃ (5%), CHCl₃ or 1M NaOH); drying (forexample, over MgSO₄, over Na₂SO₄, or in vacuo); filtering; crystallizing(for example, from EtOAc and hexane); being concentrated (for example,in vacuo); and/or purification (e.g., silica gel chromatography, flashchromatography, preparative HPLC, reverse phase-HPLC, orcrystallization).

Compounds of formula I, as well as their salts, can be prepared as shownin Scheme I:

The process comprises the step of coupling compound 1 with compound 2,where R¹, R^(2a), R^(2b), R³—R⁶, X, Z, a, and b are as defined forformula I, and P¹ is H or a suitable amino-protecting group, examples ofwhich include, t-butoxycarbonyl, trityl, benzyloxycarbonyl,9-fluorenylmethoxycarbonyl, formyl, trimethylsilyl, andt-butyldimethylsilyl. When P¹ is an amino protecting group, the processfurther comprises deprotecting the compound of formula 1, before or insitu with the coupling step.

In instances where R¹ is a group such as —OCH₃ or —OCH₂CH₃, the couplingstep may be followed by a deprotection step to provide a compound offormula I where R¹ is a group such as —OH. Thus, one method of preparingcompounds of the invention involves coupling compounds 1 and 2, with anoptional deprotection step to form a compound of formula I or apharmaceutically acceptable salt thereof.

Methods of preparing compound 1 are described in the Examples. Compound2 is generally commercially available or can be prepared usingprocedures that are known in the art.

Compounds of formula I, as well as their salts, can also be prepared asshown in Scheme II:

In this method, compound 3 is coupled with compound 4, the amine isdeprotected and the intermediate is then coupled to 5 to form a compoundof formula I or a pharmaceutically acceptable salt thereof. Methods ofpreparing compound 4 are described in the Examples. Compounds 3 and 5are generally commercially available or can be prepared using proceduresthat are known in the art.

Compounds of formula I, as well as their salts, can also be prepared asshown in Scheme III:

In the first step, compound 1 is coupled with compound 6 and compound 6is coupled to compound 7, where Y and Z react in situ to form the R³moiety. For example, when R³ is —C(O)NR²²R²³, Y is —COOH and Z isHNR²²R²³. Alternately, compound 6 is first coupled to compound 7, andthe resulting compound is then coupled with compound 1. As with SchemeI, in instances where R¹ is a group such as —OCH₃ or —OCH₂CH₃, thecoupling step may be followed by a deprotection step to provide acompound of formula I where R¹ is a group such as —OH. Thus, one methodof preparing compounds of the invention involves coupling compounds 1, 2and 3, with an optional deprotection step to form a compound of formulaI or a pharmaceutically acceptable salt thereof. Compounds 6 and 7 aregenerally commercially available or can be prepared using proceduresthat are known in the art.

Compounds of formula I, as well as their salts, can also be prepared asshown in Scheme IV:

Again, this is a standard coupling reaction between a compound offormula I, where R¹ is —OH and compound 8, to yield a compound offormula I, where R¹ is —NR⁸R⁹.

Certain intermediates described herein are believed to be novel andaccordingly, such compounds are provided as further aspects of theinvention including, for example, the compounds of formula 1, or a saltthereof:

where P¹ is H or an amino-protecting group selected fromt-butoxycarbonyl, trityl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl,formyl, trimethylsilyl, and t-butyldimethylsilyl; and R¹, R^(2a),R^(2b), R⁵, R⁶, Z, a and b are as defined for formula I. In one specificembodiment Z is —CH— and in another specific embodiment Z is —N—. Theseembodiments can be depicted as formulas 1a and 1b, respectively:

Another intermediate of the invention has formula 9 or a salt thereof:

where R^(1P) is selected from —O—P³, —NHP², and —NH(O—P⁴); where P² isan amino-protecting group selected from t-butoxycarbonyl, trityl,benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, formyl, trimethylsilyl,and t-butyldimethylsilyl; P³ is a carboxy-protecting group selected frommethyl, ethyl, t-butyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl,trimethylsilyl, t-butyldimethylsilyl, and diphenylmethyl; P⁴ is ahydroxyl-protecting group selected from —C₁₋₆alkyl, triC₁₋₆alkylsilyl,—C₁₋₆alkanoyl, benzoyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl, anddiphenylmethyl; and R^(2a), R^(2b), R³, R⁴, R⁵, R⁶, a, b, Z, and X areas defined for formula I. Another intermediate of the invention hasformula 10 or a salt thereof:

where R^(3P) is selected from —C₀₋₅alkylene-O—P⁴, —C₀₋₁alkylene-COO—P³,and phenyl substituted with —O—P⁴; P³ is a carboxy-protecting groupselected from methyl, ethyl, t-butyl, benzyl, p-methoxybenzyl,9-fluorenylmethyl, trimethylsilyl, t-butyldimethylsilyl, anddiphenylmethyl; P⁴ is a hydroxyl-protecting group selected from—C₁₋₆alkyl, triC₁₋₆alkylsilyl, —C₁₋₆alkanoyl, benzoyl, benzyl,p-methoxybenzyl, 9-fluorenylmethyl, and diphenylmethyl; and R¹, R^(2a),R^(2b), R⁴, R⁵, R⁶, a, b, Z, and X are as defined for formula I. Stillanother intermediate of the invention has formula 11 or a salt thereof:

where R^(4P) is selected from —O—P⁴; —C₁₋₂alkylene-COO—P³; and phenyl orbenzyl substituted with —COO—P³; P³ is a carboxy-protecting groupselected from methyl, ethyl, t-butyl, benzyl, p-methoxybenzyl,9-fluorenylmethyl, trimethylsilyl, t-butyldimethylsilyl, anddiphenylmethyl; P⁴ is a hydroxyl-protecting group selected from—C₁₋₆alkyl, triC₁₋₆alkylsilyl, —C₁₋₆alkanoyl, benzoyl, benzyl,p-methoxybenzyl, 9-fluorenylmethyl, and diphenylmethyl; and R¹, R^(2a),R^(2b), R³, R⁵, R⁶, a, b, Z, and X are as defined for formula I. Yetanother intermediate of the invention has formula 12 or a salt thereof:

where R^(3P) is selected from —C₀₋₅alkylene-O—P⁴, —C₀₋₁alkylene-COO—P³,and phenyl substituted with —O—P⁴; R^(4P) is selected from —O—P⁴;—C₁₋₂alkylene-COO—P³; and phenyl or benzyl substituted with —COO—P³; P³is a carboxy-protecting group selected from methyl, ethyl, t-butyl,benzyl, p-methoxybenzyl, 9-fluorenylmethyl, trimethylsilyl,t-butyldimethylsilyl, and diphenylmethyl; P⁴ is a hydroxyl-protectinggroup selected from —C₁₋₆alkyl, triC₁₋₆alkylsilyl, —C₁₋₆alkanoyl,benzoyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl, and diphenylmethyl;and R¹, R^(2a), R^(2b), R⁵, R⁶, a, b, Z, and X are as defined forformula I. Thus, another method of preparing compounds of the inventioninvolves deprotecting a compound of formula 1, 9, 10, 11, 12, or a saltthereof.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth below.

Utility

Compounds of the invention possess neprilysin (NEP) inhibition activity,that is, the compounds are able to inhibit enzyme-catalytic activity. Inanother embodiment, the compounds do not exhibit significant inhibitoryactivity of the angiotensin-converting enzyme. One measure of theability of a compound to inhibit NEP activity is the inhibition constant(pK_(i)). The pK_(i) value is the negative logarithm to base 10 of thedissociation constant (K_(i)) which is typically reported in molarunits. Compounds of the invention of particular interest are thosehaving a pK_(i) at NEP greater than or equal to 6.0, particularly thosehaving a pK_(i) greater than or equal to 7.0, and even more particularlythose having a pK_(i) greater than or equal to 8.0. In one embodiment,compounds of interest have a pK_(i) in the range of 6.0-6.9; in anotherembodiment, compounds of interest have a pK_(i) in the range of 7.0-7.9;in yet another embodiment, compounds of interest have a pK_(i) in therange of 8.0-8.9; and in still another embodiment, compounds of interesthave a pK_(i) in the range of greater than or equal to 9.0. Such valuescan be determined by techniques that are well known in the art, as wellas in the assays described herein.

Another measure of the ability of a compound to inhibit NEP activity isthe apparent inhibition constant (IC₅₀), which is the molarconcentration of compound that results in half-maximal inhibition ofsubstrate conversion by the NEP enzyme. The pIC₅₀ value is the negativelogarithm to base 10 of the IC₅₀. Compounds of the invention that are ofparticular interest, include those that exhibit a pIC₅₀ for NEP greaterthan or equal to about 5.0. Compounds of interest also include thosehaving a pIC₅₀ for NEP≧ about 6.0 or a pIC₅₀ for NEP≧ about 7.0. Inanother embodiment, compounds of interest have a pIC₅₀ for NEP withinthe range of about 7.0-10.0.

It is noted that in some cases, compounds of the invention may possessweak NEP inhibition activity. In such cases, those of skill in the artwill recognize that these compounds still have utility as researchtools.

Exemplary assays to determine properties of compounds of the invention,such as the NEP inhibiting activity, are described in the Examples andinclude by way of illustration and not limitation, assays that measureNEP inhibition (described in Assay 1). Useful secondary assays includeassays to measure ACE inhibition (also described in Assay 1) andaminopeptidase P (APP) inhibition (described in Sulpizio et al. (2005)JPET 315:1306-1313). A pharmacodynamic assay to assess the in vivoinhibitory potencies for ACE and NEP in anesthetized rats is describedin Assay 2 (see also Seymour et al. (1985) Hypertension 7 (SupplI):I-35-I-42 and Wigle et al. (1992) Can. J. Physiol. Pharmacol.70:1525-1528), where ACE inhibition is measured as the percentinhibition of the angiotensin I pressor response and NEP inhibition ismeasured as increased urinary cyclic guanosine 3′, 5′-monophosphate(cGMP) output.

There are many in vivo assays that can be used to ascertain furtherutilities of the compounds of the invention. The conscious spontaneouslyhypertensive rat (SHR) model is a renin dependent hypertension model,and is described in Assay 3. See also Intengan et al. (1999) Circulation100(22):2267-2275 and Badyal et al. (2003) Indian Journal ofPharmacology 35:349-362. The conscious desoxycorticosterone acetate-salt(DOCA-salt) rat model is a volume dependent hypertension model that isuseful for measuring NEP activity, and is described in Assay 4. See alsoTrapani et al. (1989) J Cardiovasc. Pharmacol. 14:419-424, Intengan etal. (1999) Hypertension 34(4):907-913, and Badyal et al. (2003) supra).The DOCA-salt model is particularly useful for evaluating the ability ofa test compound to reduce blood pressure as well as to measure a testcompound's ability to prevent or delay a rise in blood pressure. TheDahl salt-sensitive (DSS) hypertensive rat model is a model ofhypertension that is sensitive to dietary salt (NaCl), and is describedin Assay 5. See also Rapp (1982) Hypertension 4:753-763. The ratmonocrotaline model of pulmonary arterial hypertension described, forexample, in Kato et al. (2008) J. Cardiovasc. Pharmacol. 51(1):18-23, isa reliable predictor of clinical efficacy for the treatment of pulmonaryarterial hypertension. Heart failure animal models include the DSS ratmodel for heart failure and the aorto-caval fistula model (AV shunt),the latter of which is described, for example, in Norling et al.(1996)J. Amer. Soc. Nephrol. 7:1038-1044. Other animal models, such asthe hot plate, tail-flick and formalin tests, can be used to measure theanalgesic properties of compounds of the invention, as well as thespinal nerve ligation (SNL) model of neuropathic pain. See, for example,Malmberg et al. (1999) Current Protocols in Neuroscience 8.9.1-8.9.15.

Compounds of the invention are expected to inhibit the NEP enzyme in anyof the assays listed above, or assays of a similar nature. Thus, theaforementioned assays are useful in determining the therapeutic utilityof compounds of the invention, for example, their utility asantihypertensive agents or antidiarrheal agents. Other properties andutilities of compounds of the invention can be demonstrated using otherin vitro and in vivo assays well-known to those skilled in the art.Compounds of formula I may be active drugs as well as prodrugs. Thus,when discussing the activity of compounds of the invention, it isunderstood that any such prodrugs may not exhibit the expected activityin an assay, but are expected to exhibit the desired activity oncemetabolized.

Compounds of the invention are expected to be useful for the treatmentand/or prevention of medical conditions responsive to NEP inhibition.Thus it is expected that patients suffering from a disease or disorderthat is treated by inhibiting the NEP enzyme or by increasing the levelsof its peptide substrates, can be treated by administering atherapeutically effective amount of a compound of the invention. Forexample, by inhibiting NEP, the compounds are expected to potentiate thebiological effects of endogenous peptides that are metabolized by NEP,such as the natriuretic peptides, bombesin, bradykinins, calcitonin,endothelins, enkephalins, neurotensin, substance P and vasoactiveintestinal peptide. Thus, these compounds are expected to have otherphysiological actions, for example, on the renal, central nervous,reproductive and gastrointestinal systems.

In one embodiment of the invention, patients suffering from a disease ordisorder that is treated by inhibiting the NEP enzyme, are treated byadministering a compound of the invention that is in its active form,i.e., a compound of formula I where R¹ is selected from —OR⁷ and —NR⁸R⁹,R⁷ is H, R⁸ is H or —OH, R⁹ is H, and R^(2a), R^(2b), R³—R⁶, a, b, Z,and X are as defined for formula I.

In another embodiment, patients are treated by administering a compoundthat is metabolized in vitro to form a compound of formula I where R¹ isselected from —OR⁷ and —NR⁸R⁹, R⁷ is H, R⁸ is H or —OH, R⁹ is H, andR^(2a), R^(2b), R³—R⁶, a, b, Z, and X are as defined for formula I.

In another embodiment, patients are treated by administering a compoundof the invention that is in its prodrug form at the R¹ group, i.e., acompound of formula I where:

R¹ is —OR⁷; and R⁷ is selected from —C₁₋₈alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl,—C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃,—C₁₋₆alkylene-OC(O)R¹⁰, —C₁₋₆alkylene-NR¹²R¹³, —C₁₋₆alkylene-C(O)R³¹,—C₀₋₆alkylenemorpholinyl, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

or

R¹ is NR⁸R⁹; R⁸ is selected from —OC(O)R¹⁴, —CH₂COOH, —O-benzyl,pyridyl, and —OC(S)NR¹⁵R¹⁶; and R⁹ is H; or

R¹ is NR⁸R⁹; R⁸ is selected from —OC(O)R¹⁴, —CH₂COOH, —O-benzyl,pyridyl, and —OC(S)NR¹⁵R¹⁶; and R⁹ is —C₁₋₆alkyl or —C(O)R¹⁷;

R¹ is —NR⁸R⁹; R⁸ is selected from H or —OH; and R⁹ is —C₁₋₆alkyl or—C(O)R¹⁷;

R¹ is —OR⁷ and R^(2a) is taken together with R⁷ to form —OCR¹⁸R¹⁹—; or

R¹ is —NR⁸R⁹ and R^(2a) is taken together with R⁸ to form —OC(O)—.

and R^(2b), R¹⁰, R¹²—R¹⁷, R³¹, R³², R³—R⁶, a, b, Z, and X are as definedfor formula I. In an exemplary embodiment, patients are treated byadministering a compound of the invention that is in its prodrug form atthe R¹ group and has formula III, IV, or V.

Cardiovascular Diseases

By potentiating the effects of vasoactive peptides like the natriureticpeptides and bradykinin, compounds of the invention are expected to findutility in treating and/or preventing medical conditions such ascardiovascular diseases. See, for example, Rogues et al. (1993)Pharmacol. Rev. 45:87-146 and Dempsey et al. (2009) Amer. J. ofPathology 174(3):782-796. Cardiovascular diseases of particular interestinclude hypertension and heart failure. Hypertension includes, by way ofillustration and not limitation: primary hypertension, which is alsoreferred to as essential hypertension or idiopathic hypertension;secondary hypertension; hypertension with accompanying renal disease;severe hypertension with or without accompanying renal disease;pulmonary hypertension, including pulmonary arterial hypertension; andresistant hypertension. Heart failure includes, by way of illustrationand not limitation: congestive heart failure; acute heart failure;chronic heart failure, for example with reduced left ventricularejection fraction (also referred to as systolic heart failure) or withpreserved left ventricular ejection fraction (also referred to asdiastolic heart failure); and acute and chronic decompensated heartfailure. Thus, one embodiment of the invention relates to a method fortreating hypertension, particularly primary hypertension or pulmonaryarterial hypertension, comprising administering to a patient atherapeutically effective amount of a compound of the invention.

For treatment of primary hypertension, the therapeutically effectiveamount is typically the amount that is sufficient to lower the patient'sblood pressure. This would include both mild-to-moderate hypertensionand severe hypertension. When used to treat hypertension, the compoundmay be administered in combination with other therapeutic agents such asaldosterone antagonists, aldosterone synthase inhibitors,angiotensin-converting enzyme inhibitors and dual-actingangiotensin-converting enzymeneprilysin inhibitors,angiotensin-converting enzyme 2 (ACE2) activators and stimulators,angiotensin-II vaccines, anti-diabetic agents, anti-lipid agents,anti-thrombotic agents, AT₁ receptor antagonists and dual-acting AT₁receptor antagonistneprilysin inhibitors, β₁-adrenergic receptorantagonists, dual-acting β-adrenergic receptor antagonist/α₁-receptorantagonists, calcium channel blockers, diuretics, endothelin receptorantagonists, endothelin converting enzyme inhibitors, neprilysininhibitors, natriuretic peptides and their analogs, natriuretic peptideclearance receptor antagonists, nitric oxide donors, non-steroidalanti-inflammatory agents, phosphodiesterase inhibitors (specificallyPDE-V inhibitors), prostaglandin receptor agonists, renin inhibitors,soluble guanylate cyclase stimulators and activators, and combinationsthereof. In one particular embodiment of the invention, a compound ofthe invention is combined with an AT₁ receptor antagonist, a calciumchannel blocker, a diuretic, or a combination thereof, and used to treatprimary hypertension. In another particular embodiment of the invention,a compound of the invention is combined with an AT₁ receptor antagonist,and used to treat hypertension with accompanying renal disease. Whenused to treat resistant hypertension, the compound may be administeredin combination with other therapeutic agents such as aldosteronesynthase inhibitors.

For treatment of pulmonary arterial hypertension, the therapeuticallyeffective amount is typically the amount that is sufficient to lower thepulmonary vascular resistance. Other goals of therapy are to improve apatient's exercise capacity. For example, in a clinical setting, thetherapeutically effective amount can be the amount that improves apatient's ability to walk comfortably for a period of 6 minutes(covering a distance of approximately 20-40 meters). When used to treatpulmonary arterial hypertension the compound may be administered incombination with other therapeutic agents such as α-adrenergic receptorantagonists, β₁-adrenergic receptor antagonists, β₂-adrenergic receptoragonists, angiotensin-converting enzyme inhibitors, anticoagulants,calcium channel blockers, diuretics, endothelin receptor antagonists,PDE-V inhibitors, prostaglandin analogs, selective serotonin reuptakeinhibitors, and combinations thereof. In one particular embodiment ofthe invention, a compound of the invention is combined with a PDE-Vinhibitor or a selective serotonin reuptake inhibitor and used to treatpulmonary arterial hypertension.

Another embodiment of the invention relates to a method for treatingheart failure, in particular congestive heart failure (including bothsystolic and diastolic congestive heart failure), comprisingadministering to a patient a therapeutically effective amount of acompound of the invention. Typically, the therapeutically effectiveamount is the amount that is sufficient to lower blood pressure and/orimprove renal functions. In a clinical setting, the therapeuticallyeffective amount can be the amount that is sufficient to improve cardiachemodynamics, like for instance reduction in wedge pressure, rightatrial pressure, filling pressure, and vascular resistance. In oneembodiment, the compound is administered as an intravenous dosage form.When used to treat heart failure, the compound may be administered incombination with other therapeutic agents such as adenosine receptorantagonists, advanced glycation end product breakers, aldosteroneantagonists, AT₁ receptor antagonists, β₁-adrenergic receptorantagonists, dual-acting β-adrenergic receptor antagonist/α₁-receptorantagonists, chymase inhibitors, digoxin, diuretics, endothelinconverting enzyme (ECE) inhibitors, endothelin receptor antagonists,natriuretic peptides and their analogs, natriuretic peptide clearancereceptor antagonists, nitric oxide donors, prostaglandin analogs, PDE-Vinhibitors, soluble guanylate cyclase activators and stimulators, andvasopressin receptor antagonists. In one particular embodiment of theinvention, a compound of the invention is combined with an aldosteroneantagonist, a β₁-adrenergic receptor antagonist, an AT₁ receptorantagonist, or a diuretic, and used to treat congestive heart failure.

Diarrhea

As NEP inhibitors, compounds of the invention are expected to inhibitthe degradation of endogenous enkephalins and thus such compounds mayalso find utility for the treatment of diarrhea, including infectiousand secretory/watery diarrhea. See, for example, Baumer et al. (1992)Gut 33:753-758; Farthing (2006) Digestive Diseases 24:47-58; andMarcais-Collado (1987) Eur. J. Pharmacol. 144(2):125-132. When used totreat diarrhea, compounds of the invention may be combined with one ormore additional antidiarrheal agents.

Renal Diseases

By potentiating the effects of vasoactive peptides like the natriureticpeptides and bradykinin, compounds of the invention are expected toenhance renal function (see Chen et al. (1999) Circulation100:2443-2448; Lipkin et al. (1997) Kidney Int. 52:792-801; and Dussauleet al. (1993) Clin. Sci. 84:31-39) and find utility in treating and/orpreventing renal diseases. Renal diseases of particular interest includediabetic nephropathy, chronic kidney disease, proteinuria, andparticularly acute kidney injury or acute renal failure (see Sharkovskaet al. (2011) Clin. Lab. 57:507-515 and Newaz et al. (2010) RenalFailure 32:384-390). When used to treat renal disease, the compound maybe administered in combination with other therapeutic agents such asangiotensin-converting enzyme inhibitors, AT₁ receptor antagonists, anddiuretics.

Preventative Therapy

By potentiating the effects of the natriuretic peptides, compounds ofthe invention are also expected to be useful in preventative therapy,due to the antihypertrophic and antifibrotic effects of the natriureticpeptides (see Potter et al. (2009) Handbook of Experimental Pharmacology191:341-366), for example in preventing the progression of cardiacinsufficiency after myocardial infarction, preventing arterialrestenosis after angioplasty, preventing thickening of blood vesselwalls after vascular operations, preventing atherosclerosis, andpreventing diabetic angiopathy.

Glaucoma

By potentiating the effects of the natriuretic peptides, compounds ofthe invention are expected to be useful to treat glaucoma. See, forexample, Diestelhorst et al. (1989) International Ophthalmology12:99-101. When used to treat glaucoma, compounds of the invention maybe combined with one or more additional antiglaucoma agents.

Pain Relief

As NEP inhibitors, compounds of the invention are expected to inhibitthe degradation of endogenous enkephalins and thus such compounds mayalso find utility as analgesics. See, for example, Rogues et al. (1980)Nature 288:286-288 and Thanawala et al. (2008) Current Drug Targets9:887-894. When used to treat pain, the compounds of the invention maybe combined with one or more additional antinociceptive drugs such asaminopeptidase N or dipeptidyl peptidase III inhibitors, non-steroidalanti-inflammatory agents, monoamine reuptake inhibitors, musclerelaxants, NMDA receptor antagonists, opioid receptor agonists,5-HT_(1D) serotonin receptor agonists, and tricyclic antidepressants.

Other Utilities

Due to their NEP inhibition properties, compounds of the invention arealso expected to be useful as antitussive agents, as well as findutility in the treatment of portal hypertension associated with livercirrhosis (see Sansoe et al. (2005) J. Hepatol. 43:791-798), cancer (seeVesely (2005)J. Investigative Med. 53:360-365), depression (see Noble etal. (2007) Exp. Opin. Ther. Targets 11:145-159), menstrual disorders,preterm labor, pre-eclampsia, endometriosis, reproductive disorders (forexample, male and female infertility, polycystic ovarian syndrome,implantation failure), and male and female sexual dysfunction, includingmale erectile dysfunction and female sexual arousal disorder. Morespecifically, the compounds of the invention are expected to be usefulin treating female sexual dysfunction (see Pryde et al. (2006) J. Med.Chem. 49:4409-4424), which is often defined as a female patient'sdifficulty or inability to find satisfaction in sexual expression. Thiscovers a variety of diverse female sexual disorders including, by way ofillustration and not limitation, hypoactive sexual desire disorder,sexual arousal disorder, orgasmic disorder and sexual pain disorder.When used to treat such disorders, especially female sexual dysfunction,compounds of the invention may be combined with one or more of thefollowing secondary agents: PDE-V inhibitors, dopamine agonists,estrogen receptor agonists and/or antagonists, androgens, and estrogens.Due to their NEP inhibition properties, compounds of the invention arealso expected to have anti-inflammatory properties, and are expected tohave utility as such, particularly when used in combination withstatins.

Recent studies suggest that NEP plays a role in regulating nervefunction in insulin-deficient diabetes and diet induced obesity. Coppeyet al. (2011) Neuropharmacology 60:259-266. Therefore, due to their NEPinhibition properties, compounds of the invention are also expected tobe useful in providing protection from nerve impairment caused bydiabetes or diet induced obesity.

The amount of the compound of the invention administered per dose or thetotal amount administered per day may be predetermined or it may bedetermined on an individual patient basis by taking into considerationnumerous factors, including the nature and severity of the patient'scondition, the condition being treated, the age, weight, and generalhealth of the patient, the tolerance of the patient to the active agent,the route of administration, pharmacological considerations such as theactivity, efficacy, pharmacokinetics and toxicology profiles of thecompound and any secondary agents being administered, and the like.Treatment of a patient suffering from a disease or medical condition(such as hypertension) can begin with a predetermined dosage or a dosagedetermined by the treating physician, and will continue for a period oftime necessary to prevent, ameliorate, suppress, or alleviate thesymptoms of the disease or medical condition. Patients undergoing suchtreatment will typically be monitored on a routine basis to determinethe effectiveness of therapy. For example, in treating hypertension,blood pressure measurements may be used to determine the effectivenessof treatment. Similar indicators for other diseases and conditionsdescribed herein, are well known and are readily available to thetreating physician. Continuous monitoring by the physician will insurethat the optimal amount of the compound of the invention will beadministered at any given time, as well as facilitating thedetermination of the duration of treatment. This is of particular valuewhen secondary agents are also being administered, as their selection,dosage, and duration of therapy may also require adjustment. In thisway, the treatment regimen and dosing schedule can be adjusted over thecourse of therapy so that the lowest amount of active agent thatexhibits the desired effectiveness is administered and, further, thatadministration is continued only so long as is necessary to successfullytreat the disease or medical condition.

Research Tools

Since compounds of the invention possess NEP enzyme inhibition activity,such compounds are also useful as research tools for investigating orstudying biological systems or samples having a NEP enzyme, for exampleto study diseases where the NEP enzyme or its peptide substrates plays arole. Any suitable biological system or sample having a NEP enzyme maybe employed in such studies which may be conducted either in vitro or invivo. Representative biological systems or samples suitable for suchstudies include, but are not limited to, cells, cellular extracts,plasma membranes, tissue samples, isolated organs, mammals (such asmice, rats, guinea pigs, rabbits, dogs, pigs, humans, and so forth), andthe like, with mammals being of particular interest. In one particularembodiment of the invention, NEP enzyme activity in a mammal isinhibited by administering a NEP-inhibiting amount of a compound of theinvention. Compounds of the invention can also be used as research toolsby conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprising aNEP enzyme is typically contacted with a NEP enzyme-inhibiting amount ofa compound of the invention. After the biological system or sample isexposed to the compound, the effects of inhibiting the NEP enzyme aredetermined using conventional procedures and equipment, such as bymeasuring receptor binding in a binding assay or measuringligand-mediated changes in a functional assay. Exposure encompassescontacting cells or tissue with the compound, administering the compoundto a mammal, for example by i.p., p.o, i.v., s.c., or inhaledadministration, and so forth. This determining step can involvemeasuring a response (a quantitative analysis) or can involve making anobservation (a qualitative analysis). Measuring a response involves, forexample, determining the effects of the compound on the biologicalsystem or sample using conventional procedures and equipment, such asenzyme activity assays and measuring enzyme substrate or productmediated changes in functional assays. The assay results can be used todetermine the activity level as well as the amount of compound necessaryto achieve the desired result, that is, a NEP enzyme-inhibiting amount.Typically, the determining step will involve determining the effects ofinhibiting the NEP enzyme.

Additionally, compounds of the invention can be used as research toolsfor evaluating other chemical compounds, and thus are also useful inscreening assays to discover, for example, new compounds havingNEP-inhibiting activity. In this manner, a compound of the invention isused as a standard in an assay to allow comparison of the resultsobtained with a test compound and with compounds of the invention toidentify those test compounds that have about equal or superioractivity, if any. For example, pK_(i) data for a test compound or agroup of test compounds is compared to the pK_(i) data for a compound ofthe invention to identify those test compounds that have the desiredproperties, for example, test compounds having a pK_(i) value aboutequal or superior to a compound of the invention, if any. This aspect ofthe invention includes, as separate embodiments, both the generation ofcomparison data (using the appropriate assays) and the analysis of testdata to identify test compounds of interest. Thus, a test compound canbe evaluated in a biological assay, by a method comprising the steps of:(a) conducting a biological assay with a test compound to provide afirst assay value; (b) conducting the biological assay with a compoundof the invention to provide a second assay value; wherein step (a) isconducted either before, after or concurrently with step (b); and (c)comparing the first assay value from step (a) with the second assayvalue from step (b). Exemplary biological assays include a NEP enzymeinhibition assay.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to, oral,rectal, vaginal, nasal, inhaled, topical (including transdermal),ocular, and parenteral modes of administration. Further, the compoundsof the invention may be administered, for example orally, in multipledoses per day (for example, two, three, or four times daily), in asingle daily dose or a single weekly dose. It will be understood thatany form of the compounds of the invention, (that is, free base, freeacid, pharmaceutically acceptable salt, solvate, etc.) that is suitablefor the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the invention. The compositions may containother therapeutic and/or formulating agents if desired. When discussingcompositions, the “compound of the invention” may also be referred toherein as the “active agent,” to distinguish it from other components ofthe formulation, such as the carrier. Thus, it is understood that theterm “active agent” includes compounds of formula I as well aspharmaceutically acceptable salts, solvates and prodrugs of thatcompound.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the invention. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,such as in bulk compositions, or less than a therapeutically effectiveamount, that is, individual unit doses designed for multipleadministration to achieve a therapeutically effective amount. Typically,the composition will contain from about 0.01-95 wt % of active agent,including, from about 0.01-30 wt %, such as from about 0.01-10 wt %,with the actual amount depending upon the formulation itself, the routeof administration, the frequency of dosing, and so forth. In oneembodiment, a composition suitable for an oral dosage form, for example,may contain about 5-70 wt %, or from about 10-60 wt % of active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers and the like usingconventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable for oraladministration. Suitable compositions for oral administration may be inthe form of capsules, tablets, pills, lozenges, cachets, dragees,powders, granules; solutions or suspensions in an aqueous or non-aqueousliquid; oil-in-water or water-in-oil liquid emulsions; elixirs orsyrups; and the like; each containing a predetermined amount of theactive agent.

When intended for oral administration in a solid dosage form (capsules,tablets, pills and the like), the composition will typically comprisethe active agent and one or more pharmaceutically acceptable carriers,such as sodium citrate or dicalcium phosphate. Solid dosage forms mayalso comprise: fillers or extenders, such as starches, microcrystallinecellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid;binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; humectants, such as glycerol;disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; solution retarding agents, such as paraffin; absorptionaccelerators, such as quaternary ammonium compounds; wetting agents,such as cetyl alcohol and/or glycerol monostearate; absorbents, such askaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, optionally with one ormore of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (for example, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.Suspensions may contain suspending agents such as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminium metahydroxide, bentonite,agar-agar and tragacanth, and mixtures thereof

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, that is, each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

In another embodiment, the compositions of the invention are suitablefor inhaled administration, and will typically be in the form of anaerosol or a powder. Such compositions are generally administered usingwell-known delivery devices, such as a nebulizer, dry powder, ormetered-dose inhaler. Nebulizer devices produce a stream of highvelocity air that causes the composition to spray as a mist that iscarried into a patient's respiratory tract. An exemplary nebulizerformulation comprises the active agent dissolved in a carrier to form asolution, or micronized and combined with a carrier to form a suspensionof micronized particles of respirable size. Dry powder inhalersadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. An exemplary dry powderformulation comprises the active agent dry-blended with an excipientsuch as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Metered-doseinhalers discharge a measured amount of the active agent usingcompressed propellant gas. An exemplary metered-dose formulationcomprises a solution or suspension of the active agent in a liquefiedpropellant, such as a chlorofluorocarbon or hydrofluoroalkane. Optionalcomponents of such formulations include co-solvents, such as ethanol orpentane, and surfactants, such as sorbitan trioleate, oleic acid,lecithin, glycerin, and sodium lauryl sulfate. Such compositions aretypically prepared by adding chilled or pressurized hydrofluoroalkane toa suitable container containing the active agent, ethanol (if present)and the surfactant (if present). To prepare a suspension, the activeagent is micronized and then combined with the propellant.Alternatively, a suspension formulation can be prepared by spray dryinga coating of surfactant on micronized particles of the active agent. Theformulation is then loaded into an aerosol canister, which forms aportion of the inhaler.

Compounds of the invention can also be administered parenterally (forexample, by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agent is provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. Parenteralformulations may also contain one or more anti-oxidants, solubilizers,stabilizers, preservatives, wetting agents, emulsifiers, and dispersingagents. Surfactants, additional stabilizing agents or pH-adjustingagents (acids, bases or buffers) and anti-oxidants are particularlyuseful to provide stability to the formulation, for example, to minimizeor avoid hydrolysis of ester and amide linkages that may be present inthe compound. These formulations may be rendered sterile by use of asterile injectable medium, a sterilizing agent, filtration, irradiation,or heat. In one particular embodiment, the parenteral formulationcomprises an aqueous cyclodextrin solution as the pharmaceuticallyacceptable carrier. Suitable cyclodextrins include cyclic moleculescontaining six or more α-D-glucopyranose units linked at the 1,4positions by a linkages as in amylase, β-cyclodextrin orcycloheptaamylose. Exemplary cyclodextrins include cyclodextrinderivatives such as hydroxypropyl and sulfobutyl ether cyclodextrinssuch as hydroxypropyl-β-cyclodextrin and sulfobutyl etherβ-cyclodextrin. Exemplary buffers for such formulations includecarboxylic acid-based buffers such as citrate, lactate and maleatebuffer solutions.

Compounds of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, thecompound can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

Secondary Agents

The compounds of the invention may be useful as the sole treatment of adisease or may be combined with one or more additional therapeuticagents in order to obtain the desired therapeutic effect. Thus, in oneembodiment, pharmaceutical compositions of the invention contain otherdrugs that are co-administered with a compound of the invention. Forexample, the composition may further comprise one or more drugs (alsoreferred to as “secondary agents(s)”). Such therapeutic agents are wellknown in the art, and include adenosine receptor antagonists,α-adrenergic receptor antagonists, β₁-adrenergic receptor antagonists,β₂-adrenergic receptor agonists, dual-acting β-adrenergic receptorantagonist/α₁-receptor antagonists, advanced glycation end productbreakers, aldosterone antagonists, aldosterone synthase inhibitors,aminopeptidase N inhibitors, androgens, angiotensin-converting enzymeinhibitors and dual-acting angiotensin-converting enzymeneprilysininhibitors, angiotensin-converting enzyme 2 activators and stimulators,angiotensin-II vaccines, anticoagulants, anti-diabetic agents,antidiarrheal agents, anti-glaucoma agents, anti-lipid agents,antinociceptive agents, anti-thrombotic agents, AT₁ receptor antagonistsand dual-acting AT₁ receptor antagonistneprilysin inhibitors andmultifunctional angiotensin receptor blockers, bradykinin receptorantagonists, calcium channel blockers, chymase inhibitors, digoxin,diuretics, dopamine agonists, endothelin converting enzyme inhibitors,endothelin receptor antagonists, HMG-CoA reductase inhibitors,estrogens, estrogen receptor agonists and/or antagonists, monoaminereuptake inhibitors, muscle relaxants, natriuretic peptides and theiranalogs, natriuretic peptide clearance receptor antagonists, neprilysininhibitors, nitric oxide donors, non-steroidal anti-inflammatory agents,N-methyl d-aspartate receptor antagonists, opioid receptor agonists,phosphodiesterase inhibitors, prostaglandin analogs, prostaglandinreceptor agonists, renin inhibitors, selective serotonin reuptakeinhibitors, sodium channel blocker, soluble guanylate cyclasestimulators and activators, tricyclic antidepressants, vasopressinreceptor antagonists, and combinations thereof. Specific examples ofthese agents are detailed herein.

Accordingly, in yet another aspect of the invention, a pharmaceuticalcomposition comprises a compound of the invention, a second activeagent, and a pharmaceutically acceptable carrier. Third, fourth etc.active agents may also be included in the composition. In combinationtherapy, the amount of compound of the invention that is administered,as well as the amount of secondary agents, may be less than the amounttypically administered in monotherapy.

Compounds of the invention may be physically mixed with the secondactive agent to form a composition containing both agents; or each agentmay be present in separate and distinct compositions which areadministered to the patient simultaneously or at separate times. Forexample, a compound of the invention can be combined with a secondactive agent using conventional procedures and equipment to form acombination of active agents comprising a compound of the invention anda second active agent. Additionally, the active agents may be combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition comprising a compound of the invention, a second activeagent and a pharmaceutically acceptable carrier. In this embodiment, thecomponents of the composition are typically mixed or blended to create aphysical mixture. The physical mixture is then administered in atherapeutically effective amount using any of the routes describedherein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention, ranging anywhere from concurrent withadministration of the compound of the invention to about 24 hourspost-dose. This is also referred to as sequential administration. Thus,a compound of the invention can be orally administered simultaneously orsequentially with another active agent using two tablets, with onetablet for each active agent, where sequential may mean beingadministered immediately after administration of the compound of theinvention or at some predetermined time later (for example, one hourlater or three hours later). It is also contemplated that the secondaryagent may be administered more than 24 hours after administration of thecompound of the invention. Alternatively, the combination may beadministered by different routes of administration, that is, one orallyand the other by inhalation.

In one embodiment, the kit comprises a first dosage form comprising acompound of the invention and at least one additional dosage formcomprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc.) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount such that they are typically administered in an amountthat produces a therapeutically beneficial effect when co-administeredwith a compound of the invention. The secondary agent can be in the formof a pharmaceutically acceptable salt, solvate, optically purestereoisomer, and so forth. The secondary agent may also be in the formof a prodrug, for example, a compound having a carboxylic acid groupthat has been esterified. Thus, secondary agents listed herein areintended to include all such forms, and are commercially available orcan be prepared using conventional procedures and reagents.

In one embodiment, compounds of the invention are administered incombination with an adenosine receptor antagonist, representativeexamples of which include, but are not limited to, naxifylline,rolofylline, SLV-320, theophylline, and tonapofylline.

In one embodiment, compounds of the invention are administered incombination with an α-adrenergic receptor antagonist, representativeexamples of which include, but are not limited to, doxazosin, prazosin,tamsulosin, and terazosin.

Compounds of the invention may also be administered in combination witha β₁-adrenergic receptor antagonist (“β₁-blockers”). Representativeβ₁-blockers include, but are not limited to, acebutolol, alprenolol,amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol,bisoprolol, bopindolol, bucindolol, bucumolol, bufetolol, bufuralol,bunitrolol, bupranolol, bubridine, butofilolol, carazolol, carteolol,carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol,esmolol, indenolol, labetolol, levobunolol, mepindolol, metipranolol,metoprolol such as metoprolol succinate and metoprolol tartrate,moprolol, nadolol, nadoxolol, nebivalol, nipradilol, oxprenolol,penbutolol, perbutolol, pindolol, practolol, pronethalol, propranolol,sotalol, sufinalol, talindol, tertatolol, tilisolol, timolol,toliprolol, xibenolol, and combinations thereof. In one particularembodiment, the β₁-antagonist is selected from atenolol, bisoprolol,metoprolol, propranolol, sotalol, and combinations thereof. Typically,the β₁-blocker will be administered in an amount sufficient to providefrom about 2-900 mg per dose.

In one embodiment, compounds of the invention are administered incombination with a β₂-adrenergic receptor agonist, representativeexamples of which include, but are not limited to, albuterol,bitolterol, fenoterol, formoterol, indacaterol, isoetharine,levalbuterol, metaproterenol, pirbuterol, salbutamol, salmefamol,salmeterol, terbutaline, vilanterol, and the like Typically, theβ₂-adrenergic receptor agonist will be administered in an amountsufficient to provide from about 0.05-500 μg per dose.

In one embodiment, compounds of the invention are administered incombination with an advanced glycation end product (AGE) breaker,examples of which include, by way of illustration and not limitation,alagebrium (or ALT-711), and TRC4149.

In another embodiment, compounds of the invention are administered incombination with an aldosterone antagonist, representative examples ofwhich include, but are not limited to, eplerenone, spironolactone, andcombinations thereof. Typically, the aldosterone antagonist will beadministered in an amount sufficient to provide from about 5-300 mg perday.

In one embodiment, compounds of the invention are administered incombination with an aminopeptidase N or dipeptidyl peptidase IIIinhibitor, examples of which include, by way of illustration and notlimitation, bestatin and PC18 (2-amino-4-methylsulfonyl butane thiol,methionine thiol).

Compounds of the invention can also be administered in combination withan angiotensin-converting enzyme (ACE) inhibitor. Representative ACEinhibitors include, but are not limited to, accupril, alacepril,benazepril, benazeprilat, captopril, ceranapril, cilazapril, delapril,enalapril, enalaprilat, fosinopril, fosinoprilat, imidapril, lisinopril,moexipril, monopril,moveltipril, pentopril, perindopril, quinapril,quinaprilat, ramipril, ramiprilat, saralasin acetate, spirapril,temocapril, trandolapril, zofenopril, and combinations thereof.

In a particular embodiment, the ACE inhibitor is selected from:benazepril, captopril, enalapril, lisinopril, ramipril, and combinationsthereof. Typically, the ACE inhibitor will be administered in an amountsufficient to provide from about 1-150 mg per day. In anotherembodiment, compounds of the invention are administered in combinationwith a dual-acting angiotensin-converting enzymeneprilysin (ACENEP)inhibitor, examples of which include, but are not limited to: AVE-0848((4S,7S,12bR)-7-[3-methyl-2(S)-sulfanylbutyramido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]-benzazepine-4-carboxylicacid); AVE-7688 (ilepatril) and its parent compound; BMS-182657(2-[2-oxo-3 (S)-[3-phenyl-2(S)-sulfanylpropionamido]-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]acetic acid); CGS-35601(N-[1-[4-methyl-2(S)-sulfanylpentanamido]cyclopentylcarbonyl]-L-tryptophan);fasidotril; fasidotrilate; enalaprilat; ER-32935((3R,6S,9aR)-6-[3(S)-methyl-2(S)-sulfanylpentanamido]-5-oxoperhydrothiazolo[3,2-a]azepine-3-carboxylicacid); gempatrilat; MDL-101264((4S,7S,12bR)-7-[2(S)-(2-morpholinoacetylthio)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); MDL-101287([4S-[4α,7α(R*),12bβ]]-7[2-(carboxymethyl)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylicacid); omapatrilat; RB-105 (N-[2(5)-(mercaptomethyl)-3(R)-phenylbutyl]-L-alanine); sampatrilat; SA-898((2R,4R)—N-[2-(2-hydroxyphenyl)-3-(3-mercaptopropionyl)thiazolidin-4-ylcarbonyl]-L-phenylalanine);Sch-50690(N-[1(S)-carboxy-2-[N2-(methanesulfonyl)-L-lysylamino]ethyl]-L-yalyl-L-tyrosine);and combinations thereof, may also be included. In one particularembodiment, the ACENEP inhibitor is selected from: AVE-7688,enalaprilat, fasidotril, fasidotrilate, omapatrilat, sampatrilat, andcombinations thereof

In one embodiment, compounds of the invention are administered incombination with an angiotensin-converting enzyme 2 (ACE2) activator orstimulator.

In one embodiment, compounds of the invention are administered incombination with an angiotensin-II vaccine, examples of which include,but are not limited to ATR12181 and CYT006-AngQb.

In one embodiment, compounds of the invention are administered incombination with an anticoagulant, representative examples of whichinclude, but are not limited to: coumarins such as warfarin; heparin;and direct thrombin inhibitors such as argatroban, bivalirudin,dabigatran, and lepirudin.

In yet another embodiment, compounds of the invention are administeredin combination with an anti-diabetic agent. Representative anti-diabeticagents include injectable drugs as well as orally effective drugs, andcombinations thereof. Examples of injectable drugs include, but are notlimited to, insulin and insulin derivatives. Examples of orallyeffective drugs include, but are not limited to: biguanides such asmetformin; glucagon antagonists; α-glucosidase inhibitors such asacarbose and miglitol; dipeptidyl peptidase IV inhibitors (DPP-IVinhibitors) such as alogliptin, denagliptin, linagliptin, saxagliptin,sitagliptin, and vildagliptin; meglitinides such as repaglinide;oxadiazolidinediones; sulfonylureas such as chlorpropamide, glimepiride,glipizide, glyburide, and tolazamide; thiazolidinediones such aspioglitazone and rosiglitazone; and combinations thereof.

In another embodiment, compounds of the invention are administered incombination with antidiarrheal treatments. Representative treatmentoptions include, but are not limited to, oral rehydration solutions(ORS), loperamide, diphenoxylate, and bismuth subsalicylate.

In yet another embodiment, a compound of the invention is administeredin combination with an anti-glaucoma agent. Representative anti-glaucomaagents include, but are not limited to: α-adrenergic agonists such asbrimonidine; β₁-adrenergic receptor antagonists; topical β₁-blockerssuch as betaxolol, levobunolol, and timolol; carbonic anhydraseinhibitors such as acetazolamide, brinzolamide, or dorzolamide;cholinergic agonists such as cevimeline and DMXB-anabaseine; epinephrinecompounds; miotics such as pilocarpine; and prostaglandin analogs.

In yet another embodiment, compounds of the invention are administeredin combination with an anti-lipid agent. Representative anti-lipidagents include, but are not limited to: cholesteryl ester transferprotein inhibitors (CETPs) such as anacetrapib, dalcetrapib, andtorcetrapib; statins such as atorvastatin, fluvastatin, lovastatin,pravastatin, rosuvastatin and simvastatin; and combinations thereof.

-   -   In one embodiment, compounds of the invention are administered        in combination with an anti-thrombotic agent. Representative        anti-thrombotic agents include, but are not limited to: aspirin;        anti-platelet agents such as clopidogrel, prasugrel, and        ticlopidine; heparin, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an AT₁ receptor antagonist, also known as angiotensinII type 1 receptor blockers (ARBs). Representative ARBs include, but arenot limited to, abitesartan, azilsartan (e.g., azilsartan medoxomil),benzyllosartan, candesartan, candesartan cilexetil, elisartan,embusartan, enoltasosartan, eprosartan, EXP3174, fonsartan, forasartan,glycyllosartan, irbesartan, isoteoline, losartan, medoximil,milfasartan, olmesartan (e.g., olmesartan medoxomil), opomisartan,pratosartan, ripisartan, saprisartan, saralasin, sarmesin, TAK-591,tasosartan, telmisartan, valsartan, zolasartan, and combinationsthereof. In a particular embodiment, the ARB is selected from azilsartanmedoxomil, candesartan cilexetil, eprosartan, irbesartan, losartan,olmesartan medoxomil, saprisartan, tasosartan, telmisartan, valsartan,and combinations thereof. Exemplary salts and/or prodrugs includecandesartan cilexetil, eprosartan mesylate, losartan potassium salt, andolmesartan medoxomil. Typically, the ARB will be administered in anamount sufficient to provide from about 4-600 mg per dose, withexemplary daily dosages ranging from 20-320 mg per day.

Compounds of the invention may also be administered in combination witha dual-acting agent, such as an AT₁ receptor antagonistneprilysininhibitor (ARBNEP) inhibitor, examples of which include, but are notlimited to, compounds described in U.S. Publication Nos. 20080269305 and20090023228, both to Allegretti et al. filed on Apr. 23, 2008, such asthe compound,4′-{2-ethoxy-4-ethyl-5-[((S)-2-mercapto-4-methylpentanoylamino)-methyl]imidazol-1-ylmethyl}-3′-fluorobiphenyl-2-carboxylicacid.

Compounds of the invention may also be administered in combination withmultifunctional angiotensin receptor blockers as described in Kurtz &Klein (2009) Hypertension Research 32:826-834.

In one embodiment, compounds of the invention are administered incombination with a bradykinin receptor antagonist, for example,icatibant (HOE-140). It is expected that this combination therapy maypresent the advantage of preventing angioedema or other unwantedconsequences of elevated bradykinin levels.

In one embodiment, compounds of the invention are administered incombination with a calcium channel blocker. Representative calciumchannel blockers include, but are not limited to, amlodipine, anipamil,aranipine, barnidipine, bencyclane, benidipine, bepridil, clentiazem,cilnidipine, cinnarizine, diltiazem, efonidipine, elgodipine, etafenone,felodipine, fendiline, flunarizine, gallopamil, isradipine, lacidipine,lercanidipine, lidoflazine, lomerizine, manidipine, mibefradil,nicardipine, nifedipine, niguldipine, niludipine, nilvadipine,nimodipine, nisoldipine, nitrendipine, nivaldipine, perhexiline,prenylamine, ryosidine, semotiadil, terodiline, tiapamil, verapamil, andcombinations thereof. In a particular embodiment, the calcium channelblocker is selected from amlodipine, bepridil, diltiazem, felodipine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, ryosidine, verapamil, andcombinations thereof. Typically, the calcium channel blocker will beadministered in an amount sufficient to provide from about 2-500 mg perdose.

In one embodiment, compounds of the invention are administered incombination with a chymase inhibitor, such as TPC-806 and2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-heptyl]acetami de (NK3201).

In one embodiment, compounds of the invention are administered incombination with a diuretic. Representative diuretics include, but arenot limited to: carbonic anhydrase inhibitors such as acetazolamide anddichlorphenamide; loop diuretics, which include sulfonamide derivativessuch as acetazolamide, ambuside, azosemide, bumetanide, butazolamide,chloraminophenamide, clofenamide, clopamide, clorexolone, disulfamide,ethoxzolamide, furosemide, mefruside, methazolamide, piretanide,torsemide, tripamide, and xipamide, as well as non-sulfonamide diureticssuch as ethacrynic acid and other phenoxyacetic acid compounds such astienilic acid, indacrinone and quincarbate; osmotic diuretics such asmannitol; potassium-sparing diuretics, which include aldosteroneantagonists such as spironolactone, and Na⁺ channel inhibitors such asamiloride and triamterene; thiazide and thiazide-like diuretics such asalthiazide, bendroflumethiazide, benzylhydrochlorothiazide,benzthiazide, buthiazide, chlorthalidone,chlorothiazide,cyclopenthiazide, cyclothiazide, epithiazide, ethiazide,fenquizone, flumethiazide, hydrochlorothiazide, hydroflumethiazide,indapamide, methylclothiazide, meticrane, metolazone, paraflutizide,polythiazide, quinethazone, teclothiazide, and trichloromethiazide; andcombinations thereof. In a particular embodiment, the diuretic isselected from amiloride, bumetanide, chlorothiazide, chlorthalidone,dichlorphenamide, ethacrynic acid, furosemide, hydrochlorothiazide,hydroflumethiazide, indapamide, methylclothiazide, metolazone,torsemide, triamterene, and combinations thereof. The diuretic will beadministered in an amount sufficient to provide from about 5-50 mg perday, more typically 6-25 mg per day, with common dosages being 6.25 mg,12.5 mg or 25 mg per day.

Compounds of the invention may also be administered in combination withan endothelin converting enzyme (ECE) inhibitor, examples of whichinclude, but are not limited to, phosphoramidon, CGS 26303, andcombinations thereof

In a particular embodiment, compounds of the invention are administeredin combination with an endothelin receptor antagonist. Representativeendothelin receptor antagonists include, but are not limited to:selective endothelin receptor antagonists that affect endothelin Areceptors, such as avosentan, ambrisentan, atrasentan, BQ-123,clazosentan, darusentan, sitaxentan, and zibotentan; and dual endothelinreceptor antagonists that affect both endothelin A and B receptors, suchas bosentan, macitentan, tezosentan.

In yet another embodiment, a compound of the invention is administeredin combination with one or more HMG-CoA reductase inhibitors, which arealso known as statins. Representative statins include, but are notlimited to, atorvastatin, fluvastatin, lovastatin, pitavastatin,pravastatin, rosuvastatin and simvastatin.

In one embodiment, compounds of the invention are administered incombination with a monoamine reuptake inhibitor, examples of whichinclude, by way of illustration and not limitation, norepinephrinereuptake inhibitors such as atomoxetine, buproprion and the buproprionmetabolite hydroxybuproprion, maprotiline, reboxetine, and viloxazine;selective serotonin reuptake inhibitors (SSRIs) such as citalopram andthe citalopram metabolite desmethylcitalopram, dapoxetine, escitalopram(e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethylmetabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate),paroxetine, sertraline and the sertraline metabolite demethylsertraline;dual serotonin-norepinephrine reuptake inhibitors (SNRIs) such asbicifadine, duloxetine, milnacipran, nefazodone, and venlafaxine; andcombinations thereof.

In another embodiment, compounds of the invention are administered incombination with a muscle relaxant, examples of which include, but arenot limited to: carisoprodol, chlorzoxazone, cyclobenzaprine,diflunisal, metaxalone, methocarbamol, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a natriuretic peptide or analog, examples of whichinclude but are not limited to: carperitide, CD-NP (Nile Therapeutics),CU-NP, nesiritide, PL-3994 (Palatin Technologies, Inc.), ularitide,cenderitide, and compounds described in Ogawa et al (2004) J. Biol.Chem. 279:28625-31. These compounds are also referred to as natriureticpeptide receptor-A (NPR-A) agonists. In another embodiment, compounds ofthe invention are administered in combination with a natriuretic peptideclearance receptor (NPR-C) antagonist such as SC-46542, cANF (4-23), andAP-811 (Veale (2000) Bioorg Med Chem Lett 10:1949-52). For example,AP-811 has shown synergy when combined with the NEP inhibitor, thiorphan(Wegner (1995) Clin. Exper. Hypert. 17:861-876).

In another embodiment, compounds of the invention are administered incombination with a neprilysin (NEP) inhibitor. Representative NEPinhibitors include, but are not limited to: AHU-377; candoxatril;candoxatrilat; dexecadotril((+)-N-[2(R)-(acetylthiomethyl)-3-phenylpropionyl]glycine benzyl ester);CGS-24128(3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-24592((S)-3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionicacid); CGS-25155 (N-[9(R)-(acetylthiomethyl)-10-oxo-1-azacyclodecan-2(S)-ylcarbonyl]-4(R)-hydroxy-L-proline benzyl ester); 3-(1-carbamoylcyclohexyl)propionicacid derivatives described in WO 2006/027680 to Hepworth et al. (PfizerInc.); JMV-390-1(2(R)-benzyl-3-(N-hydroxycarbamoyl)propionyl-L-isoleucyl-L-leucine);ecadotril; phosphoramidon; retrothiorphan; RU-42827(2-(mercaptomethyl)-N-(4-pyridinyl)benzenepropionamide); RU-44004(N-(4-morpholinyl)-3-phenyl-2-(sulfanylmethyl)propionamide); SCH-32615((S)—N—[N-(1-carboxy-2-phenylethyl)-L-phenylalanyl]-β-alanine) and itsprodrug SCH-34826((S)—N—[N-[1-[[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine);sialorphin; SCH-42495(N-[2(S)-(acetylsulfanylmethyl)-3-(2-methylphenyl)propionyl]-L-methionineethyl ester); spinorphin; SQ-28132(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]leucine); SQ-28603(N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-β-alanine); SQ-29072(7-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid);thiorphan and its prodrug racecadotril; UK-69578(cis-4-[[[1-[2-carboxy-3-(2-methoxyethoxyl)propyl]cyclopentyl]carbonyl]amino]cyclohexanecarboxylicacid); UK-447,841(2-{1-[3-(4-chlorophenyl)propylcarbamoyl]-cyclopentylmethyl}-4-methoxybutyricacid); UK-505,749((R)-2-methyl-3-{1-[3-(2-methylbenzothiazol-6-yl)propylcarbamoyl]cyclopentyl}propionicacid); 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid and 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoicacid ethyl ester (WO 2007056546);daglutril[(3S,27?)-3-{1-[2′-(ethoxycarbonyl)-4′-phenylbutyl]-cyclopentan-1-carbonylamino}-2,3,4,5-tetrahydro-2-oxo-1H-1-benzazepine-1-aceticacid] described in WO 2007/106708 to Khder et al. (Novartis AG); andcombinations thereof. In a particular embodiment, the NEP inhibitor isselected from AHU-377, candoxatril, candoxatrilat, CGS-24128,phosphoramidon, SCH-32615, SCH-34826, SQ-28603, thiorphan, andcombinations thereof. In a particular embodiment, the NEP inhibitor is acompound such as daglutril or CGS-26303([N-[2-(biphenyl-4-yl)-1(S)-(1H-tetrazol-5-yl)ethyl]amino]methylphosphonicacid), which have activity both as inhibitors of the endothelinconverting enzyme (ECE) and of NEP. Other dual acting ECENEP compoundscan also be used. The NEP inhibitor will be administered in an amountsufficient to provide from about 20-800 mg per day, with typical dailydosages ranging from 50-700 mg per day, more commonly 100-600 or 100-300mg per day.

In one embodiment, compounds of the invention are administered incombination with a nitric oxide donor, examples of which include, butare not limited to nicorandil; organic nitrates such as pentaerythritoltetranitrate; and sydnonimines such as linsidomine and molsidomine.

In yet another embodiment, compounds of the invention are administeredin combination with a non-steroidal anti-inflammatory agent (NSAID).Representative NSAIDs include, but are not limited to: acemetacin,acetyl salicylic acid, alclofenac, alminoprofen, amfenac, amiprilose,aloxiprin, anirolac, apazone, azapropazone, benorilate, benoxaprofen,bezpiperylon, broperamole, bucloxic acid, carprofen, clidanac,diclofenac, diflunisal, diftalone, enolicam, etodolac, etoricoxib,fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone,flufenamic acid, flufenisal, fluprofen, flurbiprofen, furofenac,ibufenac, ibuprofen, indomethacin, indoprofen, isoxepac, isoxicam,ketoprofen, ketorolac, lofemizole, lornoxicam, meclofenamate,meclofenamic acid, mefenamic acid, meloxicam, mesalamine, miroprofen,mofebutazone, nabumetone, naproxen, niflumic acid, oxaprozin, oxpinac,oxyphenbutazone, phenylbutazone, piroxicam, pirprofen, pranoprofen,salsalate, sudoxicam, sulfasalazine, sulindac, suprofen, tenoxicam,tiopinac, tiaprofenic acid, tioxaprofen, tolfenamic acid, tolmetin,triflumidate, zidometacin, zomepirac, and combinations thereof. In aparticular embodiment, the NSAID is selected from etodolac,flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meloxicam,naproxen, oxaprozin, piroxicam, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with an N-methyl d-aspartate (NMDA) receptor antagonist,examples of which include, by way of illustration and not limitation,including amantadine, dextromethorphan, dextropropoxyphene, ketamine,ketobemidone, memantine, methadone, and so forth.

In still another embodiment, compounds of the invention are administeredin combination with an opioid receptor agonist (also referred to asopioid analgesics). Representative opioid receptor agonists include, butare not limited to: buprenorphine, butorphanol, codeine, dihydrocodeine,fentanyl, hydrocodone, hydromorphone, levallorphan, levorphanol,meperidine, methadone, morphine, nalbuphine, nalmefene, nalorphine,naloxone, naltrexone, nalorphine, oxycodone, oxymorphone, pentazocine,propoxyphene, tramadol, and combinations thereof. In certainembodiments, the opioid receptor agonist is selected from codeine,dihydrocodeine, hydrocodone, hydromorphone, morphine, oxycodone,oxymorphone, tramadol, and combinations thereof.

In a particular embodiment, compounds of the invention are administeredin combination with a phosphodiesterase (PDE) inhibitor, particularly aPDE-V inhibitor. Representative PDE-V inhibitors include, but are notlimited to, avanafil, lodenafil, mirodenafil, sildenafil (Revatio®),tadalafil (Adcirca®), vardenafil (Levitra®), and udenafil.

In another embodiment, compounds of the invention are administered incombination with a prostaglandin analog (also referred to as prostanoidsor prostacyclin analogs). Representative prostaglandin analogs include,but are not limited to, beraprost sodium, bimatoprost, epoprostenol,iloprost, latanoprost, tafluprost, travoprost, and treprostinil, withbimatoprost, latanoprost, and tafluprost being of particular interest.

In yet another embodiment, compounds of the invention are administeredin combination with a prostaglandin receptor agonist, examples of whichinclude, but are not limited to, bimatoprost, latanoprost, travoprost,and so forth.

Compounds of the invention may also be administered in combination witha renin inhibitor, examples of which include, but are not limited to,aliskiren, enalkiren, remikiren, and combinations thereof

In another embodiment, compounds of the invention are administered incombination with a selective serotonin reuptake inhibitor (SSRI).Representative SSRIs include, but are not limited to: citalopram and thecitalopram metabolite desmethylcitalopram, dapoxetine, escitalopram(e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethylmetabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate),paroxetine, sertraline and the sertraline metabolite demethylsertraline,and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a 5-HT_(1D) serotonin receptor agonist, examples ofwhich include, by way of illustration and not limitation, triptans suchas almotriptan, avitriptan, eletriptan, frovatriptan, naratriptanrizatriptan, sumatriptan, and zolmitriptan.

In one embodiment, compounds of the invention are administered incombination with a sodium channel blocker, examples of which include, byway of illustration and not limitation, carbamazepine, fosphenytoin,lamotrignine, lidocaine, mexiletine,oxcarbazepine, phenytoin, andcombinations thereof.

In one embodiment, compounds of the invention are administered incombination with a soluble guanylate cyclase stimulator or activator,examples of which include, but are not limited to ataciguat, riociguat,and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a tricyclic antidepressant (TCA), examples of whichinclude, by way of illustration and not limitation, amitriptyline,amitriptylinoxide, butriptyline, clomipramine, demexiptiline,desipramine, dibenzepin, dimetacrine, dosulepin, doxepin, imipramine,imipraminoxide, lofepramine, melitracen, metapramine, nitroxazepine,nortriptyline, noxiptiline, pipofezine, propizepine, protriptyline,quinupramine, and combinations thereof.

In one embodiment, compounds of the invention are administered incombination with a vasopressin receptor antagonist, examples of whichinclude, by way of illustration and not limitation, conivaptan andtolvaptan.

Combined secondary therapeutic agents may also be helpful in furthercombination therapy with compounds of the invention. For example,compounds of the invention can be combined with a diuretic and an ARB,or a calcium channel blocker and an ARB, or a diuretic and an ACEinhibitor, or a calcium channel blocker and a statin. Specific examplesinclude, a combination of the ACE inhibitor enalapril (in the maleatesalt form) and the diuretic hydrochlorothiazide, which is sold under themark Vaseretic®, or a combination of the calcium channel blockeramlodipine (in the besylate salt form) and the ARB olmesartan (in themedoxomil prodrug form), or a combination of a calcium channel blockerand a statin, all may also be used with the compounds of the invention.Other therapeutic agents such as α₂-adrenergic receptor agonists andvasopressin receptor antagonists may also be helpful in combinationtherapy. Exemplary α₂-adrenergic receptor agonists include clonidine,dexmedetomidine, and guanfacine.

The following formulations illustrate representative pharmaceuticalcompositions of the invention.

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), 440 g spray-dried lactose and 10 gmagnesium stearate are thoroughly blended. The resulting composition isthen loaded into hard gelatin capsules (500 mg of composition percapsule). Alternately, a compound of the invention (20 mg) is thoroughlyblended with starch (89 mg), microcrystalline cellulose (89 mg) andmagnesium stearate (2 mg). The mixture is then passed through a No. 45mesh U.S. sieve and loaded into a hard gelatin capsule (200 mg ofcomposition per capsule).

Alternately, a compound of the invention (30 g), a secondary agent (20g), 440 g spray-dried lactose and 10 g magnesium stearate are thoroughlyblended, and processed as described above.

Exemplary Gelatin Capsule Formulation for Oral Administration

A compound of the invention (100 mg) is thoroughly blended withpolyoxyethylene sorbitan monooleate (50 mg) and starch powder (250 mg).The mixture is then loaded into a gelatin capsule (400 mg of compositionper capsule). Alternately, a compound of the invention (70 mg) and asecondary agent (30 mg) are thoroughly blended with polyoxyethylenesorbitan monooleate (50 mg) and starch powder (250 mg), and theresulting mixture loaded into a gelatin capsule (400 mg of compositionper capsule).

Alternately, a compound of the invention (40 mg) is thoroughly blendedwith microcrystalline cellulose (Avicel PH 103; 259.2 mg) and magnesiumstearate (0.8 mg). The mixture is then loaded into a gelatin capsule(Size #1, White, Opaque) (300 mg of composition per capsule).

Exemplary Tablet Formulation for Oral Administration

A compound of the invention (10 mg), starch (45 mg) and microcrystallinecellulose (35 mg) are passed through a No. 20 mesh U.S. sieve and mixedthoroughly. The granules so produced are dried at 50-60° C. and passedthrough a No. 16 mesh U.S. sieve. A solution of polyvinylpyrrolidone (4mg as a 10% solution in sterile water) is mixed with sodiumcarboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg), and talc (1mg), and this mixture is then passed through a No. 16 mesh U.S. sieve.The sodium carboxymethyl starch, magnesium stearate and talc are thenadded to the granules. After mixing, the mixture is compressed on atablet machine to afford a tablet weighing 100 mg.

Alternately, a compound of the invention (250 mg) is thoroughly blendedwith microcrystalline cellulose (400 mg), silicon dioxide fumed (10 mg),and stearic acid (5 mg). The mixture is then compressed to form tablets(665 mg of composition per tablet).

Alternately, a compound of the invention (400 mg) is thoroughly blendedwith cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120mg), and magnesium stearate (5 mg). The mixture is then compressed toform a single-scored tablet (600 mg of composition per tablet).

Alternately, a compound of the invention (100 mg) is thoroughly blendedwith cornstarch (100 mg) with an aqueous solution of gelatin (20 mg).The mixture is dried and ground to a fine powder. Microcrystallinecellulose (50 mg) and magnesium stearate (5 mg) are then admixed withthe gelatin formulation, granulated and the resulting mixture compressedto form tablets (100 mg of the compound of the invention per tablet).

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of the compound of the invention per 10 mL of suspension:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 gSodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum ® K(magnesium 1.0 g aluminum silicate) Flavoring 0.035 mL Colorings 0.5 mgDistilled water q.s. to 100 mL

Exemplary Liquid Formulation for Oral Administration

A suitable liquid formulation is one with a carboxylic acid-based buffersuch as citrate, lactate and maleate buffer solutions. For example, acompound of the invention (which may be pre-mixed with DMSO) is blendedwith a 100 mM ammonium citrate buffer and the pH adjusted to pH 5, or isblended with a 100 mM citric acid solution and the pH adjusted to pH 2.Such solutions may also include a solubilizing excipient such as acyclodextrin, for example the solution may include 10 wt %hydroxypropyl-P-cyclodextrin.

Other suitable formulations include a 5% NaHCO₃ solution, with orwithout cyclodextrin.

Exemplary Injectable Formulation for Administration by Injection

A compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

Exemplary Compositions for Administration by Inhalation

A compound of the invention (0.2 mg) is micronized and then blended withlactose (25 mg). This blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a dry powder inhaler, for example.

Alternately, a micronized compound of the invention (10 g) is dispersedin a solution prepared by dissolving lecithin (0.2 g) in demineralizedwater (200 mL). The resulting suspension is spray dried and thenmicronized to form a micronized composition comprising particles havinga mean diameter less than about 1.5 μm. The micronized composition isthen loaded into metered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of the invention per dose whenadministered by the inhaler.

Alternately, a compound of the invention (25 mg) is dissolved in citratebuffered (pH 5) isotonic saline (125 mL). The mixture is stirred andsonicated until the compound is dissolved. The pH of the solution ischecked and adjusted, if necessary, to pH 5 by slowly adding aqueous 1 NNaOH. The solution is administered using a nebulizer device thatprovides about 10 μg to about 500 μg of the compound of the inventionper dose.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard, generally accepted meaning:

-   -   AcOH acetic acid    -   DCC dicyclohexylcarbodiimide    -   DCM dichloromethane or methylene chloride    -   DIPEA N,N-diisopropylethylamine    -   DMAP 4-dimethylaminopyridine    -   DMF N,N-dimethylformamide    -   EDCI N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide    -   Et₂O diethyl ether    -   EtOAc ethyl acetate    -   EtOH ethanol    -   HATU N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium        hexafluorophosphate    -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   HOAt 1-hydroxy-7-azabenzotriazole    -   HOBt 1-hydroxybenzotriazole    -   LiHMDS lithium hexamethyl disilazide    -   Mca (7-methoxycoumarin-4-yl)acyl    -   MeCN acetonitrile    -   MeOH methanol    -   Pd(dppf)₂Cl₂ 1,1-bis(diphenylphosphino) ferrocene palladium        chloride    -   PMBOH p-methoxybenzyl alcohol    -   SilicaCat®DPP-Pd silica based diphenylphosphine palladium (II)        catalyst    -   TFA trifluoroacetic acid    -   Ti(Oi-Pr)₄ titanium tetraisopropoxide    -   THF tetrahydrofuran

Unless noted otherwise, all materials, such as reagents, startingmaterials and solvents, were purchased from commercial suppliers (suchas Sigma-Aldrich, Fluka Riedel-de Haën, and the like) and were usedwithout further purification.

Reactions were run under nitrogen atmosphere, unless noted otherwise.The progress of reactions were monitored by thin layer chromatography(TLC), analytical high performance liquid chromatography (anal. HPLC),and mass spectrometry, the details of which are given in specificexamples. Solvents used in analytical HPLC were as follows: solvent Awas 98% H₂O/2% MeCN 1.0 mL/L TFA; solvent B was 90% MeCN/10% H₂O/1.0mL/L TFA.

Reactions were worked up as described specifically in each preparationor example; commonly reaction mixtures were purified by extraction andother purification methods such as temperature-, and solvent-dependentcrystallization, and precipitation. In addition, reaction mixtures wereroutinely purified by preparative HPLC, typically using Microsorb C18and Microsorb BDS column packings and conventional eluents. Progress ofreactions was typically measured by liquid chromatography massspectrometry (LCMS). Characterization of isomers were done by NuclearOverhauser effect spectroscopy (NOE). Characterization of reactionproducts was routinely carried out by mass and ¹H-NMR spectrometry. ForNMR measurement, samples were dissolved in deuterated solvent (CD₃OD,CDCl₃, or DMSO-d₆), and ¹H-NMR spectra were acquired with a VarianGemini 2000 instrument (400 MHz) under standard observation conditions.Mass spectrometric identification of compounds was typically conductedusing an electrospray ionization method (ESMS) with an AppliedBiosystems (Foster City, Calif.) model API 150 EX instrument or anAgilent (Palo Alto, Calif.) model 1200 LCMSD instrument.

Preparation 1[(S)-1-Biphenyl-4-ylmethyl-2-(2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-yl)-ethyl]-carbamicAcid t-Butyl Ester

Step 1: To a solution of(R)-3-biphenyl-4-yl-2-t-butoxycarbonylamino-propionic acid (50 g, 0.1mol), Meldrum's acid (23.3 g, 0.2 mol) and DMAP (27.8 g, 0.2 mol) inanhydrous DCM (500 mL) was added a solution of DCC (33.3 g, 0.2 mol) inanhydrous DCM (200 mL) over 1 hour at −5° C. under nitrogen. The mixturewas stirred at −5° C. for 8 hours, then refrigerated overnight, duringwhich tiny crystals of dicyclohexylurea precipitated. After filtration,the mixture was washed with 5% KHSO₄ (4×200 mL), saturated aqueous NaCl(200 mL) and dried under refrigeration with MgSO₄ overnight. Theresulting solution was evaporated to yield crude compound 1 as a lightyellow solid (68 g). LC-MS: [M+Na]: 490, [2M+Na]: 957.

Step 2: To a solution of crude compound 1 (68 g, 0.1 mol) in anhydrousDCM (1 L) was added AcOH (96.8 g, 1.6 mol) at −5° C. under nitrogen. Themixture was stirred at −5° C. for 0.5 hour, then NaBH₄ (13.9 g, 0.4 mol)was added in small portions over 1 hour. After stirring at −5° C. foranother 1 hour, saturated aqueous NaCl (300 mL) was added. The organiclayer was washed with saturated aqueous NaCl (2×300 mL) and water (2×300mL), dried over MgSO₄, filtered, and concentrated to give the crudeproduct which was further purified by chromatography (hexanes:EtOAc=5:1)to yield the title compound as a light yellow solid (46 g). LC-MS:[M+Na]: 476, [2M+Na]: 929.

Preparation 2 (R)-4-Amino-5-biphenyl-4-yl-2-hydroxy-2-methyl-pentanoicAcid Ethyl Ester

Step 1: To a solution of[(S)-1-biphenyl-4-ylmethyl-2-(2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-yl)-ethyl]-carbamicacid t-butyl ester (46 g, 0.1 mol) in tertiary butyl alcohol (100 mL)was added dimethylmethyleneimmonium iodide (46.3 g, 0.3 mol) at roomtemperature under nitrogen. The mixture was heated to 65° C. and stirredat this temperature for 16 hours. After filtration, the filtrate wasconcentrated to give the crude product which was further purified bychromatography (hexanes:EtOAc=20:1˜10:1) to yield compound 1 as a lightyellow solid (18 g). LC-MS: [M+Na]: 460, [2M+Na]: 897.

Step 2: To a solution of compound 1 (18 g, 44 mmol) in acetone (430 mL)and water (22 mL) was added Sudan Red as indicator. Ozone atmosphere wasintroduced into the mixture at 0° C. until the red color of Sudan Reddisappeared. Dimethyl sulfide (45 mL) was added and the mixture wasstirred at room temperature overnight. The mixture was then concentratedand the residue was purified by chromatography (hexanes:EtOAc=15:1˜7:1)to yield compound 2 as a light yellow solid (9.5 g). LC-MS: [M+H]: 434,[2M+H]: 845.

Step 3: To a solution of compound 2 (9.5 g, 23 mmol) in anhydrous THF(120 mL) was added a solution of methylmagnesium bromide in THF (9.2 mL,28 mmol) at −70° C. under nitrogen. The mixture was stirred at −60° C.for 3 hours and the reaction was then quenched with saturated aqueousNH₄Cl (50 mL). The organic layer was separated and dried over MgSO₄. Themixture was then concentrated and the residue was purified bychromatography (hexanes:EtOAc=10:1˜5:1) to yield compound 3 as an oil(7.9 g). LC-MS: [M+H]: 450, [2M+H]: 877.

Step 4: To a solution of compound 3 (7.9 g, 18.4 mmol) in anhydrous DCM(300 mL) was pumped HCl atmosphere at 0° C. for 6 hours. The mixture wasthen concentrated and the residue was washed with anhydrous Et₂O toyield the title compound as a white solid HCl salt (5.8 g). LC-MS:[M+H]: 364, [2M+H]: 727. ¹H NMR (300 MHz, DMSO): δ8.00-7.97 (d, 4H),7.67-7.62 (m, 6H), 7.47-7.28 (m, 8H), 6.32 (s, 1H), 6.09 (s, 1H),4.13-4.06 (m, 2H), 3.95-3.78 (m, 2H), 3.60 (s, 1H), 3.22-3.08 (m, 3H),2.95-2.65 (m, 2H), 1.99-1.79 (m, 4H), 1.30-0.87 (m, 9H).

Example 1

A.(2R,4R)-5-Biphenyl-4-yl-2-hydroxy-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)-amino]-pentanoicacid ethyl ester (R⁷═—CH₂CH₃) B.(2R,4R)-5-Biphenyl-4-yl-2-hydroxy-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)-amino]-pentanoicacid (R⁷═H)

(R)-4-Amino-5-biphenyl-4-yl-2-hydroxy-2-methyl-pentanoic acid ethylester (300 mg, 916 μmol, 1.0 eq.), 1,2,3-triazole-4-carboxylic acid (114mg, 1.0 mmol, 1.1 eq.), and DIPEA (479 μL, 2.8 mmol, 3.0 eq.) weredissolved in DMF (6.0 mL). HATU (383 mg, 1.0 mmol, 1.1 eq.) was addedand the mixture was stirred at room temperature overnight. The mixturewas split 5050 and the two solutions were concentrated. One portion waspurified by preparative HPLC (10-70% MeCN/water), followed by isomerseparation by preparative HPLC to yield compound A (27 mg, 96% purity).MS m/z [M+H]⁺ calc'd for C₂₃H₂₆N₄O₄, 423.20. found 423.2.

The remaining portion was dissolved in THF (2.0 mL), followed by theaddition of lithium hydroxide monohydrate (192 mg, 4.6 mmol) in water(2.0 mL). The resulting mixture was stirred at room temperature untilthe reaction was complete (˜70 minutes). The reaction was quenched byaddition of AcOH, then the solution was concentrated. The crude productwas purified by preparative HPLC (10-70% MeCN/water), followed by isomerseparation by preparative HPLC to yield the compound B (24.9 mg, 100%purity). MS m/z [M+H]⁺ calc'd for C₂₁H₂₂N₄O₄, 395.16. found 395.2.

Example 2

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, compoundshaving formula IIIa-1, were also prepared:

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 1 —OH H H C₂₁H₂₂N₄O₄395.16 395.2

-   1.    (2S,4R)-5-Biphenyl-4-yl-2-hydroxy-2-methyl-4-[(3H-[1,2,3]triazole-4-carbonyl)-amino]-pentanoic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 2 —OH H H C₂₁H₂₂N₄O₄395.16 395.0 3 —OH Cl H C₂₁H₂₁ClN₄O₄ 429.13 429.4

-   2.    (R)-5-Biphenyl-4-yl-2-hydroxy-2-methyl-4-[(2H-[1,2,4]triazole-3-carbonyl)-amino]-pentanoic    acid-   3.    (R)-5-Biphenyl-4-yl-4-[(5-chloro-2H-[1,2,4]triazole-3-carbonyl)-amino]-2-hydroxy-2-methyl-pentanoic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 4 —OH F H C₂₅H₂₃FN₄O₄463.17 463.0 5 —OH Cl H C₂₅H₂₃ClN₄O₄ 479.14 479.0

-   4.    (R)-5-Biphenyl-4-yl-4-[(7-fluoro-3H-benzotriazole-5-carbonyl)-amino]-2-hydroxy-2-methyl-pentanoic    acid-   5.    (R)-5-Biphenyl-4-yl-4-[(7-chloro-3H-benzotriazole-5-carbonyl)-amino]-2-hydroxy-2-methyl-pentanoic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 6 —OH H H C₂₄H₂₃N₅O₄446.18 446.4

-   6.    (2S,4R)-5-Biphenyl-4-yl-2-hydroxy-2-methyl-4-[(1H-[1,2,3]triazolo[4,5-b]pyridine-6-carbonyl)-amino]-pentanoic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 7 —OH —OH H C₂₂H₂₂N₂O₆411.15 411.2 8 —O—CH₂CH₃ —OH H C₂₄H₂₆N₂O₆ 439.18 439.4

-   7.    (R)-5-Biphenyl-4-yl-2-hydroxy-4-[(3-hydroxy-isoxazole-5-carbonyl)-amino]-2-methyl-pentanoic    acid-   8.    (R)-5-Biphenyl-4-yl-2-hydroxy-4-[(3-hydroxy-isoxazole-5-carbonyl)-amino]-2-methyl-pentanoic    acid ethyl ester

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 9 —OH —OH H C₂₃H₂₃N₃O₅422.16 422.2 10 —OH —OH H C₂₃H₂₃N₃O₅ 422.16 422.2

-   9.    (2R,4R)-5-Biphenyl-4-yl-2-hydroxy-4-[(2-hydroxy-pyrimidine-5-carbonyl)-amino]-2-methyl-pentanoic    acid-   10.    (2S,4R)-5-Biphenyl-4-yl-2-hydroxy-4-[(2-hydroxy-pyrimidine-5-carbonyl)-amino]-2-methyl-pentanoic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 11 —OH —COOH HC₂₃H₂₃N₃O₆ 438.16 438.2

-   11.    5-((1R,3S)-1-Biphenyl-4-ylmethyl-3-carboxy-3-hydroxy-butylcarbamoyl)-2H-pyrazole-3-carboxylic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 12 —OH —OH HC₂₂H₂₃N₃O₅ 410.16 410.4 13 —OH —COOH H C₂₃H₂₃N₃O₆ 438.16 438.2

-   12.    (R)-5-Biphenyl-4-yl-2-hydroxy-4-[(5-hydroxy-2H-pyrazole-3-carbonyl)-amino]-2-methyl-pentanoic    acid-   13.    5-((1R,3R)-1-Biphenyl-4-ylmethyl-3-carboxy-3-hydroxy-butylcarbamoyl)-1H-pyrazole-3-carboxylic    acid

Preparation 33-(N-Biphenyl-4-ylmethyl-N′-t-butoxycarbonyl-hydrazino)-2-hydroxy-2-methyl-propionicAcid Methyl Ester

Step 1: 4-(Bromomethyl)biphenyl (2.0 g, 8.1 mmol, 1.0 eq.) and DIPEA(1.4 mL) were dissolved in DMF (40 mL). t-Butyl carbazate (2.1 g, 16.2mmol, 2.0 eq.) was added and the mixture was stirred at room temperatureuntil the reaction was complete (several days). The mixture waspartially concentrated, and the residue was partitioned between EtOAcand saturated aqueous NaHCO₃ solution. The EtOAc layer was then driedover Na₂SO₄ and concentrated. The crude product was purified by flashchromatography (0-60% EtOAc/hexanes w/0.5% DIPEA) to yield compound 1(1.3 g).

Step 2: Compound 1 (1.3 g, 4.3 mmol, 1.0 eq.) was dissolved in isopropylalcohol (20.0 mL). Methyl 2-methylglycidate (676 μL, 1.5 eq.) was addedand the resulting mixture was heated at 85° C. until the reaction was70% complete (several days). The mixture was partitioned between EtOAcand saturated aqueous NaHCO₃ solution, then the EtOAc layer was driedover Na₂SO₄ and concentrated. The crude product was purified by flashchromatography (0-60% EtOAc/hexanes w/0.1% DIPEA) to yield the titlecompound (1.1 g)

Example 3

A.3[N-Biphenyl-4-ylmethyl-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionicacid ethyl ester (R⁷═—CH₂CH₃ B.3[N-Biphenyl-4-ylmethyl-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionicacid (R⁷═H)

3-(N-Biphenyl-4-ylmethyl-N′-t-butoxycarbonyl-hydrazino)-2-hydroxy-2-methyl-propionicacid methyl ester (400 mg, 965 μmol, 1.0 eq.) was dissolved in DCM (4.0mL). TFA (4.0 mL) was added and the mixture was stirred at roomtemperature until the reaction was complete (˜1 hour). The mixture wasthen concentrated. 3-Hydroxyisoxazole-5-carboxylic acid (137 mg, 1.1mmol, 1.1 eq.) was dissolved in DMF (4 mL), then DIPEA (504 μL), HOAt(144 mg, 1.1 mmol, 1.1 eq.) and EDCI (188 μL) were added and the mixturewas stirred for 15 minutes. To this was added the concentrated mixturefrom above in DMF (4 mL) and the resulting mixture was stirred at roomtemperature until the reaction was complete (˜overnight). The reactionmixture was split 50/50 and the two solutions were concentrated. Oneportion was purified by preparative HPLC (10-70% MeCN/water). A solutionof HCl in EtOH was prepared by the addition of acetyl chloride (427 μL)to EtOH (6 mL). 130 mg of the purified portion was then added and themixture was stirred at 50° C. until the reaction was complete (˜2.5hours). The mixture was concentrated and the crude product was purifiedby preparative HPLC (10-70% MeCN/water) to yield compound A (99.6 mg,99% purity). MS m/z [M+H]⁺ calc'd for C₂₃H₂₅N₃O₆, 440.17. found 440.4.

The remaining portion was dissolved in THF (3.0 mL), followed by theaddition of lithium hydroxide monohydrate (202 mg, 4.8 mmol) in water(3.0 mL). The resulting mixture was stirred at room temperature untilthe reaction was complete (˜45 minutes). The reaction was quenched byaddition of AcOH, then the solution was concentrated. The crude productwas purified by preparative HPLC (10-70% MeCN/water) to yield compound B(58.2 mg, 97% purity). MS m/z [M+H]⁺ calc'd for C₂₁H₂₁N₃O₆, 412.14.found 412.4.

Example 4

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, compoundshaving formula IIIb-1, were also prepared:

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 1 —OH H H C₂₀H₂₁N₅O₄396.16 396.2

-   1.    3-[N-Biphenyl-4-ylmethyl-N′-(3H-[1,2,3]triazole-4-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 2 —OH Cl HC₂₀H₂₀ClN₅O₄ 430.12 430.4

-   2.    3-[N-Biphenyl-4-ylmethyl-N′-(5-chloro-4H-[1,2,4]triazole-3-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 3 —OH —OCH₃ HC₂₂H₂₃N₃O₆ 426.16 426.4

-   3.    3-[N-Biphenyl-4-ylmethyl-N′-(3-methoxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 4 —OH —OH H C₂₂H₂₂N₄O₅423.16 423.2

-   4.    3-[N-Biphenyl-4-ylmethyl-N′-(2-hydroxy-pyrimidine-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 5 —OH —OH H C₂₁H₂₂N₄O₅411.16 411.2 6 —OH —COOH H C₂₂H₂₂N₄O₆ 439.15 439.4 7 —OH —C(O)N(CH₃)₂ HC₂₄H₂₇N₅O₅ 466.20 466.4 8 —OH

H C₂₆H₂₉N₅O₅ 492.22 492.4

-   5.    3-[N-Biphenyl-4-ylmethyl-N′-(5-hydroxy-2H-pyrazole-3-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   6.    5-[N′-Biphenyl-4-ylmethyl-N′-(2-carboxy-2-hydroxy-propyl)-hydrazinocarbonyl]-1H-pyrazole-3-carboxylic    acid-   7.    3-[N-Biphenyl-4-ylmethyl-N′-(5-dimethylcarbamoyl-2H-pyrazole-3-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   8.    3-{N-Biphenyl-4-ylmethyl-N′-[5-(pyrrolidine-1-carbonyl)-2H-pyrazole-3-carbonyl]-hydrazino}-2-hydroxy-2-methyl-propionic    acid

Preparation 43-[N′-t-Butoxycarbonyl-N-(3′-fluorobiphenyl-4-ylmethyl)-hydrazino]-2-hydroxy-2-methyl-propionicAcid Methyl Ester

Step 1: 4-Bromobenzyl bromide (2.0 g, 8.1 mmol, 1.0 eq.) and DIPEA (1.4mL) were dissolved in DMF (40.0 mL). t-Butyl carbazate (2.1 g, 16.2mmol, 2.0 eq.) was added and the mixture was stirred at room temperatureuntil the reaction was complete (˜overnight). The mixture was partiallyconcentrated, then the residue was partitioned between EtOAc andsaturated sodium bicarbonate. The EtOAc layer was then dried over Na₂SO₄and concentrated. The crude product was purified by flash chromatography(0-60% EtOAc/hexanes w/0.1% DIPEA) to yield compound 1 (1.4 g).

Step 2: Compound 1 (720 mg, 2.4 mmol, 1.0 eq.) was dissolved inisopropyl alcohol (10.0 mL). Methyl 2-methylglycidate (380 μL) was addedand the mixture was stirred at 85° C. until the reaction was complete(several days). The mixture was partitioned between EtOAc and saturatedsodium bicarbonate. The EtOAc layers were dried over Na₂SO₄ andconcentrated to yield crude compound 2 (890 mg), which was used withoutfurther purification.

Step 3: Compound 2 (642 mg, 1.5 mmol, 1.0 eq.), 3-Fluorophenylboronicacid (430 mg, 3.1 mmol, 2.0 eq.) and K₂CO₃ (637 mg, 4.6 mmol) weredissolved in a mixture of THF (12.0 mL) and water (12.0 mL). Thereaction flask was then purged with nitrogen,Tetrakis(triphenylphosphine)palladium(O) (355 mg, 307 μmol) was added,and the reaction mixture was stirred at 90° C. until the reaction wascomplete (overnight). The mixture was diluted with water then wasextracted twice with EtOAc. The EtOAc layers were then dried over Na₂SO₄and concentrated. The crude product was purified by preparative HPLC(10-80% MeCN/water) to yield the title compound (342 mg).

Example 53-[N-(3′-Fluorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionicAcid

3-[N′-t-Butoxycarbonyl-N-(3′-fluorobiphenyl-4-ylmethyl)-hydrazino]-2-hydroxy-2-methyl-propionicacid methyl ester (100 mg, 231 μmol, 1.0 eq.) was dissolved in DCM (2.0mL). TFA (2.0 mL) was added and the mixture was stirred at roomtemperature until the reaction was complete (˜45 minutes). The mixturewas then concentrated. 3-Hydroxyisoxazole-5-carboxylic acid (38.8 mg,300 μmol, 1.3 eq.) and 1 HOAt (40.9 mg, 300 μmol, 1.3 eq.) weredissolved in DMF (2 mL) of DMF, then EDCI (53.2 μL) was added followedby DIPEA (121 μL) and the mixture was stirred for 30 minutes. To thiswas added the concentrated mixture from above in DMF (2 mL) and theresulting mixture was stirred at room temperature until the reaction wascomplete (˜2.5 hours). The mixture was then dissolved in THF (2.0 mL),followed by the addition of lithium hydroxide monohydrate (97.0 mg, 2.3mmol) in water (2.0 mL. The resulting mixture was stirred at roomtemperature until the reaction was complete (˜1 hour). The reaction wasquenched by addition of AcOH, then the solution was concentrated. Thecrude product was purified by preparative HPLC (10-70% MeCN/water) toyield the title compound (43 mg; purity 99%). MS m/z [M+H]⁺ calc'd forC₂₁H₂₀FN₃O₆, 430.13. found 430.4.

Preparation 5 (R)-4-methoxybenzyl 2-methyloxirane-2-carboxylate

To a solution of (R,R) diethyl tartrate (6.2 g, 30 mmol) and Ti(Oi-Pr)₄(7.1 g, 25 mmol) in dry DCM (500 mL) was added fresh dried 4 Å molecularsieve (30 g) at room temperature. The resulting mixture was cooled to−30° C. and t-butyl hydroperoxide (5.5M in decane, 100 mL, 550 mmol) wasadded and the solution was stirred for 30 minutes at −30° C.˜−20° C.Then, a solution of 2-methyl-2-propen-1-ol (36 g, 0.5 mol) in dry DCM(100 mL) was added dropwise at −30° C.˜−20° C. After the addition, themixture was stirred at −20° C. until the reaction was complete (2 days).The mixture was filtered and the filtrate was cooled to 0° C. A solutionof Na₂SO₃ (18 g) in water (50 mL) was added dropwise to quench thereaction. The mixture was stirred for 1 hour at 0° C. and filtered. TheDCM layer was separated and the aqueous layer was extracted with DCM(5×40 mL). The combined organic solution was dried over anhydrousNa₂SO₄, concentrated at 0° C. under vacuum and purified (silica gelchromatography; hexanes to DCM to Et₂O) to yield compound 1 (28 g) as acolorless oil.

To a suspension of NaIO₄ (72.95 g, 340 mmol) and NaHCO₃ (47.9 g, 570mmol) in water (340 mL) was added compound 1 (10 g, 114 mmol) and CCl₄(220 mL)CHCl₃ (220 mL), followed by RuCl₃ (0.5 g). After the addition,the mixture was stirred until the reaction was complete (4 hours). Themixture was filtered and the filtrate was concentrated under vacuum. Theaqueous layer was acidified to pH=2-3 with concentrated HCl, thenextracted with DCM/isopropanol (3:1, 6×300 mL). The combined organicsolution was dried over anhydrous Na₂SO₄ and concentrated to yieldcompound 2 (12 g) as a colorless oil.

To a solution of compound 2 (12 g, 118 mmol) and PMBOH (16.2 g, 118mmol) in dry DCM (200 mL) was added EDCI (27 g, 140 mmol) and DMAP (4.3g, 35.4 mmol) at 0° C. After the addition, the mixture was stirred atroom temperature until the reaction was complete (overnight). Themixture was washed with water (200 mL), saturated KHSO₄ (200 mL),saturated aqueous NaCl (100 mL), and dried over Na₂So₄, thenconcentrated under vacuum. The crude product was purified (silica gelchromatography; hexanes:EtOAc=20:1) to yield the title compound (18 g)as a colorless oil (PMB=p-methoxybenzyl).

Preparation 6(R)-3-[N-(4-Bromobenzyl)hydrazino]-2-hydroxy-2-methyl-propionic AcidEthyl Ester

To a solution of t-butyl 2-(4-bromobenzyl)hydrazinecarboxylate (9.5 g,31.5 mmol) in isopropyl alcohol (170 mL) was added (R)-4-methoxybenzyl2-methyloxirane-2-carboxylate (7 g, 31.5 mmol) under nitrogen. Themixture was heated under reflux for 4 days, then cooled to roomtemperature. The mixture was concentrated under vacuum and the residuewas purified by silica gel chromatography (hexanes:EtOAc, 4:1) to yieldcompound 1 (7.9 g) as a white solid. LC-MS: [M+Na]⁺: 545/547.

To a stirred solution of compound 1 (7.9 g, 15.0 mmol) in EtOH (80 mL)was added LiOH.H₂O (0.8 g, 19.5 mmol). The mixture was stirred at 55° C.for 3 hours, then cooled to room temperature. The mixture wasconcentrated, diluted with water (80 mL), and washed with EtOAc (40mL×3). The aqueous solution was acidified with 1N HCl to pH=3, then,extracted with EtOAc (80 mL×3). The combined organic layers were washedwith saturated aqueous NaCl (80 mL), dried over anhydrous Na₂SO₄, andconcentrated under vacuum to yield compound 2 (5.5 g) as a white solid.LC-MS: [M+Na]⁺: 425/427.

Compound 2 (6.5 g, 16.1 mmol) was dissolved in HCl/EtOH (1M, 50 mL), andthe mixture was stirred overnight at room temperature. The resultingmixture was neutralized with aqueous Na₂CO₃ (1N) to pH=8, then,concentrated under vacuum and extracted with EtOAc (50 mL×3). Thecombined organic layers were acidified with 1N HCl to pH=6, thenextracted with water (50 mL×3). The combined aqueous layers were washedwith EtOAc (50 mL×3) and concentrated to yield the title compound (2.0g) as a light yellow solid HCl salt. LC-MS: [M+H]⁺: 331/333. ¹H NMR:(CD₃OD) 1.114 (t, J=6.9 Hz, 3H), 1.344 (s, 3H), 2.844-2.902 (m, 1H),3.151 (d, J=13.8 Hz, 1H), 3.834-3.943 (m, 2H), 4.019-4.068 (m, 1H),4.118-4.202 (m, 1H), 7.253-7.280 (m, 2H), 7.545-7.573 (m, 2H).

Preparation 7(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-hydrazino]-2-hydroxy-2-methyl-propionicAcid Ethyl Ester

To a solution of t-butyl 2-(4-bromobenzyl)hydrazinecarboxylate (50 g,166 mmol) in 1,4-dioxane (600 mL) was added5-chloro-2-fluorophenylboronic acid (28.95 g, 166 mmol) and Pd(dppf)₂Cl₂(6.9 g). The mixture was stirred at room temperature under nitrogen for10 minutes, then K₂CO₃ (45.9 g, 332 mmol) in water (180 mL) was added.The mixture was stirred at 80° C. for 5 hours, then cooled to roomtemperature and concentrated under vacuum. The residue was extractedwith EtOAc (3×200 mL) and the combined organic layers were washed withsaturated aqueous NaCl (200 mL), dried over anhydrous Na₂SO₄, andconcentrated under vacuum. The residue was purified by silica gelchromatography (hexanes/EtOAc, 15:1˜4:1) to yield compound 1 (38 g) asan off-white solid. LC-MS: [M+H]⁺: 351; [M−tBu+H]⁺: 295.

To a solution of compound 1 (15.6 g, 45 mmol) in isopropyl alcohol (240mL) was added (R)-4-methoxybenzyl 2-methyloxirane-2-carboxylate (10 g,45 mmol) under nitrogen. The mixture was stirred at reflux for 4 days,then cooled to room temperature. The mixture was concentrated and theresidue was purified by silica gel chromatography (hexanes/EtOAc, 3:1)to yield compound 2 (18 g) as a white solid. LC-MS: [M+H]⁺: 573;[M−tBu+H]⁺: 517.

To a stirred solution of compound 2 (34 g, 59.3 mmol) in EtOH (300 mL)was added LiOH.H₂O (3 g, 71.2 mmol). The mixture was stirred at 55° C.for 3 hours, then cooled to room temperature. The mixture wasconcentrated, diluted with water (300 mL), and washed with EtOAc (3×100mL). The aqueous solution was acidified with 1N HCl to pH=3, thenextracted with EtOAc (3×150 mL). The combined organic layers were washedwith saturated aqueous NaCl (150 mL), dried over anhydrous Na₂SO₄, andconcentrated under vacuum to yield compound 3 (22.8 g) as a white solid.LC-MS: [M+Na]⁺: 475.

Compound 3 (22.8 g, 50.3 mmol) was dissolved in HCl/EtOH (1M, 250 mL),and the mixture was stirred overnight at room temperature. The resultingmixture was neutralized with aqueous Na₂CO₃ (2N) to pH=7, which was thenconcentrated under vacuum and extracted with EtOAc (3×100 mL). Thecombined organic layers were acidified with 1N HCl to pH=4-5, thenextracted with water (3×100 mL). The combined aqueous layers were washedwith EtOAc (3×50 mL) and concentrated to yield the title compound (8.7g) as a white solid HCl salt. LC-MS: [M+H]⁺: 381. ¹H NMR: (CD₃OD) 1.103(t, J=7.2 Hz, 3H), 1.406 (s, 3H), 2.941 (d, J=14.1 Hz, 1H), 3.293 (d,J=14.1 Hz, 1H), 3.908-3.980 (m, 2H), 4.148-4.886 (m, 2H), 7.205-7.268(m, 1H), 7.374-7.615 (m, 6H).

Example 6 A.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionicacid

3-Hydroxyisoxazole-5-carboxylic acid (23.3 mg, 181 μmol) and HATU (68.9mg, 181 μmol) were combined in DMF (1 mL), and the resulting mixture wasstirred for 5 minutes at room temperature. DIPEA (66 μL, 377 μmol) and(R)-3-[N-(4-bromo-benzyl)-hydrazino]-2-hydroxy-2-methyl-propionic acidethyl ester (50 mg, 0.2 mmol) were added and the resulting mixture wasstirred for 20 minutes. The mixture was the evaporated under reducedpressure, then combined with 5-chloro-2-fluorophenylboronic acid (47.4mg, 272 μmol), K₂CO₃ (62.6 mg, 453 μmol), EtOH (0.7 mL, 10 mmol), andwater (0.2 mL). SilicaCat®DPP-Pd (0.28 mmol/g loading; 53.9 mg, 15 μmol)was added and the resulting mixture was microwaved at 120° C. for 10minutes. The mixture was filtered, and 1 M aqueous LiOH (604 μL, 604μmol) was added. The mixture was stirred for 1 hour, then concentratedand purified by preparative HPLC to yield the title compound (33 mg, 94%purity). MS m/z [M+H]⁺ calc'd for C₂₁H₁₉ClFN₃O₆, 464.09. found 464.1.

B.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionicacid ethyl ester

3-Hydroxyisoxazole-5-carboxylic acid (132 mg, 1.0 mmol) and HATU (389mg, 1.0 mmol) were combined in DMF (3.7 mL), and the resulting mixturewas stirred for 10 minutes at room temperature. DIPEA (343 μL, 2.0 mmol)and(R)-3-[N-(5′-chloro-2′-fluorobiphenyl-4-ylmethyl)-hydrazino]-2-hydroxy-2-methyl-propionicacid ethyl ester (300 mg, 0.8 mmol) were added and the resulting mixturewas stirred for 30 minutes. LCMS showed desired product. The mixture wasthe evaporated under reduced pressure to yield the title compound (210mg, 100% purity). MS m/z [M+H]⁺ calc'd for C₂₃H₂₃ClFN₃O₆, 492.13. found492.2.

C.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionicacid heptyl ester

(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionicacid ethyl ester (100 mg, 0.2 mmol) was dissolved in 1-heptanol (6 g, 50mmol), followed by the addition of 4 M HCl in dioxane (1 mL, 4 mmol).The resulting mixture was stirred at room temperature for 8 hours, thenevaporated under reduced pressure and purified (Interchim reverse phasechromatography column) The pure fractions were lyophilized to yield thetitle compound (27 mg, 95% purity). MS m/z [M+H]⁺ calc'd forC₂₈H₃₃ClFN₃O₆, 562.20. found 562.2.

D.(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionicacid isobutyl ester

(R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionicacid ethyl ester (100 mg, 0.2 mmol) was dissolved in isobutyl alcohol (6mL, 60 mmol), followed by the addition of 4 M HCl in dioxane (1 mL, 4mmol). The resulting mixture was stirred at room temperature for 8hours, then evaporated under reduced pressure and purified bypreparative HPLC. The clean fractions were combined and lyophilized toyield the title compound (52 mg, 100% purity). MS m/z [M+H]⁺ calc'd forC₂₅H₂₇ClFN₃O₆, 520.16. found 520.2.

Example 7

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, compoundshaving formula IIIb-2, were also prepared:

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ b R⁶ Formula calcd found 1 H H H 1 3′-ClC₂₀H₂₀ClN₅O₄ 430.12 430.4

-   1.    (R)-3-[N-(3′-Chlorobiphenyl-4-ylmethyl)-N′-(3H-[1,2,3]triazole-4-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ b R⁶ Formula calcd found 2 H H —OH 2 3′-ClC₂₀H₂₀ClN₅O₅ 446.12 446.4 3 H H —OH 2 2′-F, 5′-Cl C₂₀H₁₉ClFN₅O₅ 464.11464.2 4 —(CH₂)₆—CH₃ H —OH 2 2′-F, 5′-Cl C₂₇H₃₃ClFN₅O₅ 562.22 563.1 5—CH—(CH₃)₂ H —OH 2 2′-F, 5′-Cl C₂₃H₂₅ClFN₅O₅ 506.15 506.3 6—CH₂—CH—(CH₃)₂ H —OH 2 2′-F, 5′-Cl C₂₄H₂₇ClFN₅O₅ 520.17 520.3 7 —CH₂—CH₃H —OH 2 2′-F, 5′-Cl C₂₂H₂₃ClFN₅O₅ 492.14 492.3

-   2.    (R)-3-[N-(3′-Chlorobiphenyl-4-ylmethyl)-N′-(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   3.    (R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   4.    (R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid heptyl ester-   5.    (R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid isopropyl ester-   6.    (R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid isobutyl ester-   7.    (R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid ethyl ester

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ b R⁶ Formula calcd found  8 H

H 1 3′-Cl C₂₅H₂₃ClN₆O₄ 507.15 507.2  9 H —C(O)—N(CH₃)₂ H 1 3′-ClC₂₄H₂₆ClN₅O₅ 500.16 500.2 10 H —COOH H 1 3′-Cl C₂₂H₂₁ClN₄O₆ 473.12 473.4

-   8.    (R)-3-[N-(3′-Chlorobiphenyl-4-ylmethyl)-N′-(5-pyrazin-2-yl-2H-pyrazole-3-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   9.    (R)-3-[N-(3′-Chlorobiphenyl-4-ylmethyl)-N′-(5-dimethylcarbamoyl-2H-pyrazole-3-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   10.    5-[N′—((R)-2-Carboxy-2-hydroxy-propyl)-N′-(3′-chlorobiphenyl-4-ylmethyl)-hydrazinocarbonyl]-1H-pyrazole-3-carboxylic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ b R⁶ Formula calcd found 11 H —OH H 13′-Cl C₂₁H₂₀ClN₃O₆ 446.10 446.4 12 H —OH H 2 2′-CH₃, 5′-Cl C₂₂H₂₂ClN₃O₆460.12 460.0 13 H —OH H 2 3′,5′-diCl C₂₁H₁₉Cl₂N₃O₆ 480.07 481.1 14 H —OHH 1 2′-OCH₃ C₂₂H₂₃N₃O₇ 442.15 442.2 15 H —OH H 1 2′-Cl C₂₁H₂₀ClN₃O₆446.10 446.0 16 H —OH H 1 2′-OH C₂₁H₂₁N₃O₇ 428.14 428.2 17 H —OH H 12′-F C₂₁H₂₀FN₃O₆ 430.13 430.2 18 H —OH H 2 2′,5′-diF C₂₁H₁₉F₂N₃O₆ 448.12448.2 19 H —OH H 2 2′,5′-diCl C₂₁H₁₉Cl₂N₃O₆ 480.07 480.0 20 H —OH H 13′-F C₂₁H₂₀FN₃O₆ 430.13 430.2

-   11.    (R)-3-[N-(3′-Chlorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   12.    (R)-3-[N-(5′-Chloro-2′-methyl-biphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   13.    (R)-3-[N-(3′,5′-Dichlorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   14.    (R)-2-Hydroxy-3-[N′-(3-hydroxy-isoxazole-5-carbonyl)-N-(2′-methoxy-biphenyl-4-ylmethyl)-hydrazino]-2-methyl-propionic    acid-   15.    (R)-3-[N-(2′-Chlorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   16.    (R)-2-Hydroxy-3-[N-(2′-hydroxy-biphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-methyl-propionic    acid-   17.    (R)-3-[N-(2′-Fluorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   18.    (R)-3-[N-(2′,5′-Difluorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   19.    (R)-3-[N-(2′,5′-Dichlorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   20.    (R)-3-[N-(3′-Fluorobiphenyl-4-ylmethyl)-N′-(3-hydroxy-isoxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ b R⁶ Formula calcd found 21 H ═O H 1 3′-ClC₂₁H₂₀ClN₃O₆ 446.10 446.1 22 H ═O H 2 2′-F, 5′-Cl C₂₁H₁₉ClFN₃O₆ 464.09464.0

-   21.    (R)-3-[N-(3′-Chlorobiphenyl-4-ylmethyl)-N′-(2-oxo-2,3-dihydro-oxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   22.    (R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(2-oxo-2,3-dihydro-oxazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ b R⁶ Formula calcd found 23 H absent H 22′-F, 5′-Cl C₁₉H₁₈ClFN₆O₄ 449.11 449.2 24 H absent H 2 2′-CH₃, 5′-ClC₂₀H₂₁ClN₆O₄ 445.13 445.4

-   23.    (R)-3-[N-(5′-Chloro-2′-fluorobiphenyl-4-ylmethyl)-N′-(1H-tetrazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid-   24.    (R)-3-[N-(5′-Chloro-2′-methyl-biphenyl-4-ylmethyl)-N′-(1H-tetrazole-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ b R⁶ Formula calcd found 25 H —OH H 13′-Cl C₂₂H₂₁ClN₄O₅ 457.12 457.2

-   25.    (R)-3-[N-(3′-Chlorobiphenyl-4-ylmethyl)-N′-(2-hydroxy-pyrimidine-5-carbonyl)-hydrazino]-2-hydroxy-2-methyl-propionic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ b R⁶ Formula calcd found 26 H —COOH H 13′-Cl C₂₃H₂₁ClN₄O₆ 485.12 485.2

-   26.    6-[N′—((R)-2-Carboxy-2-hydroxy-propyl)-N′-(3′-chlorobiphenyl-4-ylmethyl)-hydrazinocarbonyl]-pyrimidine-4-carboxylic    acid

Preparation 8 (R)-4-Amino-5-biphenyl-4-yl-2,2-dimethyl-pentanoic AcidEthyl Ester

Step 1: A solution of[(S)-1-biphenyl-4-ylmethyl-2-(2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-yl)-ethyl]-carbamicacid t-butyl ester (46 g, 0.1 mol) in anhydrous toluene (300 mL) wasrefluxed for 3 hours under nitrogen. After evaporation of the solvent,the residue was purified by chromatography (hexanes:EtOAc=10:1) to yieldcompound 1 as a light yellow solid (27 g). LC-MS: [M+Na]: 374, [2M+Na]:725.

Step 2: To a solution of compound 1 (6.2 g, 17.6 mmol) in anhydrous THF(100 mL) was added a solution of LiHMDS in THF (39 mL, 39 mmol) at −78°C. under nitrogen. The mixture was stirred at −78° C. for 2 hours, andthen methyl iodide (7.5 g, 53 mmol) was added. After stirring for 0.5hour at −78° C., the mixture was warmed to room temperature and stirredat room temperature for 3 hours. After the mixture cooled to −10° C.,the reaction was quenched with saturated aqueous NH₄Cl (100 mL) andextracted with EtOAc (100 mL×4). The combined organic layers were washedwith saturated aqueous NaCl (300 mL), dried over MgSO₄, filtered, andconcentrated to yield the crude product which was further purified bychromatography (hexanes:EtOAc=10:1) to yield compound 2 as a lightyellow solid (5.7 g). LC-MS: [M+Na]: 402, [2M+Na]: 781.

Step 3: To a solution of compound 2 (5.7 g, 15 mmol) in acetone (120 mL)was added 1 M NaOH (60 mL, 60 mmol) at −5° C. under nitrogen. Themixture was warmed to room temperature and stirred at room temperaturefor 20 hours. The mixture was concentrated and the residue was dilutedwith water (250 mL) and washed with EtOAc (150 mL). The pH of theaqueous layer was adjusted to 2 with 6 M HCl at 0° C., and the solid wasfiltrated and dried in vacuo to yield the crude compound 3 as a whitesolid (5 g). LC-MS: [M+Na]: 420, [2M+Na]: 817.

Step 4: To a solution of crude compound 3 (5 g, 12.7 mmol) in anhydrousEtOH (300 mL) was added SOCl₂ (13.4 mL, 190 mmol) at −30° C. undernitrogen. The mixture was warmed to room temperature and stirred for 20hours at room temperature. The mixture was concentrated, and the residuewas washed with anhydrous Et₂O to yield the title compound as a whitesolid HCl salt (3.7 g). LC-MS: [M+H]: 326, [2M+H]: 651. ¹H NMR (300 MHz,DMSO): δ7.86 (s, 3H), 7.67-7.64 (m, 4H), 7.49-7.33 (m, 5H), 4.09-3.97(m, 2H), 3.42 (m, 1H), 2.90-2.80 (m, 2H), 1.88-1.84 (m, 2H), 1.17-1.12(m, 9H).

Example 8

A.(R)-5-Biphenyl-4-yl-4-[(3-hydroxy-isoxazole-5-carbonyl)-amino]-2,2-dimethyl-pentanoicacid (R⁷═H)

3-Hydroxyisoxazole-5-carboxylic acid (396 mg, 307 μmol, 1.0 eq.), EDCI(54.4 μL, 307 μmol, 1.0 eq.) and HOBt (41.5 mg, 307 μmol, 1.0 eq.) werecombined in DMF (0.2 mL) and stirred for 5 minutes at room temperature.(R)-4-Amino-5-biphenyl-4-yl-2,2-dimethyl-pentanoic acid ethyl ester (100mg, 307 μmmol, 1.0 eq.) was added and the resulting mixture was stirredfor 30 minutes. 1 M LiOH in water (1.2 mL) in THF (3 mL) was added andthe mixture was maintained at 60° C. for 2 days. A small amount of MeOHwas added to speed up the reaction. The reaction was then quenched withAcOH and the product was purified by preparative HPLC to yield the titlecompound (25 mg, 95% purity. MS m/z [M+H]⁺ calc'd for C₂₃H₂₄N₂O₅,409.17. found 409.4.

B.(R)-5-Biphenyl-4-yl-4-[(3-hydroxy-isoxazole-5-carbonyl)-amino]-2,2-dimethyl-pentanoicacid ethyl ester (R⁷═—CH₂CH₂)

(R)-4-Amino-5-biphenyl-4-yl-2,2-dimethyl-pentanoic acid ethyl ester(72.1 mg, 221 μmol, 1.0 eq.), 1-butanol (5 mL) and 4 M of HCl in1,4-dioxane (2 mL) were combined and stirred at 65° C. overnight. Tothis was added a mixture of 3-hydroxyisoxazole-5-carboxylic acid (28.6mg, 221 μmol, 1.0 eq.), EDCI (39.2 μL, 221 μmol, 1.0 eq.) and HOBt (29.9mg, 221 μmol, 1.0 eq.) that had been combined in DMF (0.2 mL) andstirred for 5 minutes at room temperature. The resulting mixture wasstirred for 30 minutes. The reaction was then quenched with AcOH and theproduct was purified by preparative HPLC to yield the title compound (50mg, 95% purity). MS m/z [M+H]⁺ calc'd for C₂₅H₂₈N₂O₅, 437.20. found437.4.

C.(R)-5-Biphenyl-4-yl-4-[(3-hydroxy-isoxazole-5-carbonyl)-amino]-2,2-dimethyl-pentanoicacid butyl ester (R⁷═—(CH₂₃CH₃)

(R)-4-Amino-5-biphenyl-4-yl-2,2-dimethyl-pentanoic acid ethyl ester(72.1 mg, 221 μmol, 1.0 eq.) was combined with dry butyl alcohol (5 mL)and 4N HCl in dioxane (1 mL) and stirred at 60° C. overnight. Thesolvent where evaporated and azeotroped with toluene. To this was addeda mixture of 3-Hydroxyisoxazole-5-carboxylic acid (28.6 mg, 221 μmol,1.0 eq.), EDCI (39.2 μL, 1.0 eq.) and HOBt (29.9 mg, 221 μmol, 1.0 eq.)that had been combined in DMF (0.2 mL) and stirred for 5 minutes at roomtemperature. The resulting mixture was stirred for 30 minutes. Thereaction was quenched with AcOH and the product was purified bypreparative HPLC and lyophilized to yield the title compound (40 mg, 95%purity). MS m/z [M+H]⁺ calc'd for C₂₇H₃₂N₂O₅, 465.23. found 465.4.

Example 9

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, compoundshaving formula IVα-1, were also prepared:

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 1 —OH H H C₂₂H₂₄N₄O₃393.19 393.0 2 —OCH₂CH₃ H H C₂₄H₂₈N₄O₃ 421.22 421.2

-   1.    (R)-5-Biphenyl-4-yl-2,2-dimethyl-4-[(3H-[1,2,3]triazole-4-carbonyl)-amino]-pentanoic    acid-   2.    (R)-5-Biphenyl-4-yl-2,2-dimethyl-4-[(3H-[1,2,3]triazole-4-carbonyl)-amino]-pentanoic    acid ethyl ester

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 3 —OH —OH H C₂₂H₂₄N₄O₄409.18 409.4 4 —OH H H C₂₂H₂₄N₄O₃ 393.19 393.2

-   3.    (R)-5-Biphenyl-4-yl-4-[(5-hydroxy-2H-[1,2,4]triazole-3-carbonyl)-amino]-2,2-dimethyl-pentanoic    acid-   4.    (R)-5-Biphenyl-4-yl-2,2-dimethyl-4-[(2H-[1,2,4]triazole-3-carbonyl)-amino]-pentanoic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 5 —OH —OH H C₂₄H₂₅N₃O₄420.18 420.4

-   5.    (R)-5-Biphenyl-4-yl-4-[(2-hydroxy-pyrimidine-5-carbonyl)-amino]-2,2-dimethyl-pentanoic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found  6 —OH —COOH HC₂₄H₂₅N₃O₅ 436.18 436.2  7 —OCH₂CH₃ —COOH H C₂₆H₂₉N₃O₅ 464.21 464.2  8—OH

H C₂₈H₃₂N₄O₅ 505.24 505.2  9 —OH

H C₂₈H₃₂N₄O₅ 505.24 505.2 10 —OH

H C₂₉H₃₄N₄O₅ 519.25 519.2 11 —OH

H C₂₉H₃₂N₄O₆ 533.23 533.2 12 —OH —C(O)—NH(CH₃) H C₂₅H₂₈N₄O₄ 449.21 449.213 —OH —C(O)N(CH₃)₂ H C₂₆H₃₀N₄O₄ 463.23 463.2 14 —OH—C(O)N(CH₃)—CH₂—CH(CH₃)₂ H C₂₉H₃₆N₄O₄ 505.27 505.2 15 —OH—C(O)N(CH₃)—(CH₂)₂OCH₃ H C₂₈H₃₄N₄O₅ 507.25 507.2 16 —OCH₂CH₃—C(O)N(CH₃)—(CH₂)₂OCH₃ H C₃₀H₃₈N₄O₅ 535.28 535.2 17 —OH—C(O)NH—(CH₂)₂-imidazole H C₂₉H₃₂N₆O₄ 529.25 529.2 18 —OH —COCH₃ HC₂₅H₂₇N₃O₄ 434.20 434.2

-   6.    5-((R)-1-Biphenyl-4-ylmethyl-3-carboxy-3-methyl-butylcarbamoyl)-1H-pyrazole-3-carboxylic    acid-   7.    5-((R)-1-Biphenyl-4-ylmethyl-3-ethoxycarbonyl-3-methyl-butylcarbamoyl)-1H-pyrazole-3-carboxylic    acid-   8.    (R)-5-Biphenyl-4-yl-2,2-dimethyl-4-{[5-(morpholine-4-carbonyl)-2H-pyrazole-3-carbonyl]-amino}-pentanoic    acid-   9.    (R)-5-Biphenyl-4-yl-4-{[5-3-hydroxy-pyrrolidine-1-carbonyl)-2H-pyrazole-3-carbonyl]-amino}-2,2-dimethyl-pentanoic    acid-   10.    (R)-5-Biphenyl-4-yl-4-{[5-(4-hydroxy-piperidine-1-carbonyl)-2H-pyrazole-3-carbonyl]-amino}-2,2-dimethyl-pentanoic    acid-   11.    (S)-1-[5-((R)-1-Biphenyl-4-ylmethyl-3-carboxy-3-methyl-butylcarbamoyl)-1H-pyrazole-3-carbonyl]-pyrrolidine-2-carboxylic    acid-   12.    (R)-5-Biphenyl-4-yl-2,2-dimethyl-4-[(5-methylcarbamoyl-2H-pyrazole-3-carbonyl)-amino]-pentanoic    acid-   13.    (R)-5-Biphenyl-4-yl-4-[(5-dimethylcarbamoyl-2H-pyrazole-3-carbonyl)-amino]-2,2-dimethyl-pentanoic    acid-   14.    (R)-5-Biphenyl-4-yl-4-{[5-(isobutyl-methyl-carbamoyl)-2H-pyrazole-3-carbonyl]-amino}-2,2-dimethyl-pentanoic    acid-   15.    (R)-5-Biphenyl-4-yl-4-({5-[(2-methoxy-ethyl)-methyl-carbamoyl]-2H-pyrazole-3-carbonyl}-amino)-2,2-dimethyl-pentanoic    acid-   16.    (R)-5-Biphenyl-4-yl-4-({5-[(2-methoxy-ethyl)-methyl-carbamoyl]-2H-pyrazole-3-carbonyl}-amino)-2,2-dimethyl-pentanoic    acid ethyl ester-   17.    (R)-5-Biphenyl-4-yl-4-({5-[2-(1H-imidazol-4-yl)-ethylcarbamoyl]-2H-pyrazole-3-carbonyl}-amino)-2,2-dimethyl-pentanoic    acid-   18.    (R)-4-[(5-Acetyl-2H-pyrazole-3-carbonyl)-amino]-5-biphenyl-4-yl-2,2-dimethyl-pentanoic    acid

Preparation 91-((R)-2-Amino-3-biphenyl-4-yl-propyl)-cyclopropanecarboxylic Acid

Into a flask containing BOC-D-4,4′-biphenylalanine (11.3 g, 33.1 mmol,1.0 eq.), 4-dimethylaminopyridine (6.5 g, 53.0 mmol, 1.6 eq.),2,2-dimethyl-1,3-dioxane-4,6-dione (5.3 g, 36.4 mmol, 1.1 eq.) in DCM(100 mL) was added 1 M of DCC in DCM (38.1 mL) at 0° C. over 30 minutes.The mixture was maintained at 0° C. for 6 hours and the resultingprecipitate was filtered off. The filtrate was washed with aqueous 10%KHSO₄ (2×50 mL) then dried. The solution was acidified with AcOH (20 mL)at 0° C. and sodium borohydride (3.1 g, 82.7 mmol, 2.5 eq.) was addedover 30 minutes in 3 portions. The mixture was maintained at 0° C. for 3hours, washed with water and dried, then concentrated under vacuum. Thecrude material was purified by chromatography (0-40% EtOAc/hexanesgradient). Eschenmoser's salt (15.9 g, 86.0 mmol) in t-butyl alcohol (70mL) was added and the resulting mixture was stirred at 65° C. overnight.The mixture was concentrated and Et₂O (10 mL) was added. The organicsolution was then washed with saturated aqueous NaHCO₃ (10 mL) and 10%KHSO₄ (10 mL). The organic solution was dried over Na₂SO₄ andconcentrated. The crude product was purified by chromatography (0-40%EtOAc/hexanes gradient) to yield compound 1 (3.3 g).

Trimethylsufoxonium iodide (2.0 g, 9.2 mmol, 1.0 eq.) in dimethylsulfoxide (50 mL) was combined with NaH (366 mg, 9.2 mmol, 1.1 eq.) andstirred for 15 minutes at room temperature. To this was added compound 1(3.6 g, 8.3 mmol, 1.0 eq) dissolved dimethyl sulfoxide (50 mL). Theresulting mixture was stirred at room temperature overnight. Thesolution was mixed with saturated aqueous NaCl (50 mL) and extractedwith EtOAc (3×10 mL), and the organic layer was washed with saturatedaqueous NaCl (2×50 mL) and dried over anhydrous Na₂SO₄. Afterevaporation of the solvent, the crude reaction was purified bychromatography (0-40% EtOAc/hexanes gradient) to yield compound 2,1-((R)-3-biphenyl-4-yl-2-t-butoxycarbonylaminopropyl)cyclopropanecarboxylicacid t-butyl ester. TFA (200 μL) and DCM (500 μL) were added and theresulting mixture was stirred for 30 minutes. The solvent was evaporatedunder vacuum and azeotroped with toluene (2×) to obtain the titlecompound.

Example 10

A.5-[(R)-2-Biphenyl-4-yl-1-(1-carboxy-cyclopropylmethyl)-ethylcarbamoyl]-2H-pyrazole-3-carboxylicacid (R⁷═H)

3,5-Pyrazoledicarboxylic acid (35.2 mg, 226 μmol, 1.0 eq.), DIPEA (126μL) and HATU (85.9 mg, 226 μmol, 1.0 eq.) and DCM (5 mL) were combinedand stirred for 5 minutes at room temperature.1-((R)-2-Amino-3-biphenyl-4-yl-propyl)-cyclopropanecarboxylic acid (86.7mg, 294 μmol, 2.3 eq,) and DIPEA (0.5 mL) in DCM (5 mL) was added, andthe resulting mixture was stirred for 1 hour. The reaction was quenchedwith saturated aqueous NH₄Cl and the product was extracted with DCM,dried and evaporated. The resulting product was combined with AcOH (1.5mL) and purified with preparative HPLC to yield the title compound (21.4mg; 93% purity). MS m/z [M+H]⁺ calc'd for C₂₄H₂₃N₃O₅, 434.16. found433.5.

B.5-[(R)-1-Biphenyl-4-ylmethyl-2-(1-butoxycarbonyl-cyclopropyl)-ethylcarbamoyl]-1H-pyrazole-3-carboxylicacid (R⁷═—(CH₂)₃CH₃)

1-((R)-3-Biphenyl-4-yl-2-t-butoxycarbonylaminopropyl)cyclopropanecarboxylicacid t-butyl ester (1.0 g, 2.2 mmol, 1.0 eq.), 1-butanol (5 mL) and 4 Mof HCl in 1,4-dioxane (2 mL) were combined and stirred at 65° C.overnight. To this was added a mixture of 3,5-pyrazoledicarboxylic acid(34.6 mg, 221 μmol, 1.0 eq.), EDCI (39.2 μL, 221 μmol, 1.0 eq.) and HOBt(29.9 mg, 221 μmol, 1.0 eq.) that had been combined in DMF (0.2 mL) andstirred for 5 minutes at room temperature. The resulting mixture wasstirred for 30 minutes. The reaction was then quenched with ACOH and theproduct was purified by preparative HPLC and lyophilized to yield thetitle compound (29 mg, 95% purity). MS m/z [M+H]⁺ calc'd for C₂₈H₃₁N₃O₅,490.23. found 490.6.

Note that as explained herein, compounds such as this can exist in atautomer form, for example, as5-[(R)-1-biphenyl-4-ylmethyl-2-(1-butoxycarbonyl-cyclopropyl)-ethylcarbamoyl]-2H-pyrazole-3-carboxylicacid.

Example 11

Following the procedures described in the examples herein, andsubstituting the appropriate starting materials and reagents, compoundshaving formula Vα-1, were also prepared:

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 1 —OH H H C₂₂H₂₂N₄O₃391.17 391.4 2 —OCH₂CH₃ H H C₂₄H₂₆N₄O₃ 419.20 419.2

-   1.    1-{(R)-3-Biphenyl-4-yl-2-[(3H-[1,2,3]triazole-4-carbonyl)-amino]-propyl}-cyclopropanecarboxylic    acid-   2.    1-{(R)-3-Biphenyl-4-yl-2-[(3H-[1,2,3]triazole-4-carbonyl)-amino]-propyl}-cyclopropanecarboxylic    acid ethyl ester

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 3 —OH —OH H C₂₂H₂₂N₄O₄407.16 407.4 4 —O(CH₂)₃CH₃ —OH H C₂₆H₃₀N₄O₄ 463.23 463.4

-   3.    1-{(R)-3-Biphenyl-4-yl-2-[(5-hydroxy-2H-[1,2,4]triazole-3-carbonyl)-amino]-propyl}-cyclopropanecarboxylic    acid-   4.    1-{(R)-3-Biphenyl-4-yl-2-[(5-hydroxy-2H-[1,2,4]triazole-3-carbonyl)-amino]-propyl}-cyclopropanecarboxylic    acid butyl ester

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 5 —OH —OH H C₂₃H₂₂N₂O₅407.15 407.4 6 —OCH₂CH₃ —OH H C₂₅H₂₆N₂O₅ 435.18 435.4 7 —O(CH₂)₃CH₃ —OHH C₂₇H₃₀N₂O₅ 463.22 463.4

-   5.    1-{(R)-3-Biphenyl-4-yl-2-[(3-hydroxy-isoxazole-5-carbonyl)-amino]-propyl}-cyclopropanecarboxylic    acid-   6.    1-{(R)-3-Biphenyl-4-yl-2-[(3-hydroxy-isoxazole-5-carbonyl)-amino]-propyl}-cyclopropanecarboxylic    acid ethyl ester-   7.    1-{(R)-3-Biphenyl-4-yl-2-[(3-hydroxy-isoxazole-5-carbonyl)-amino]-propyl}-cyclopropanecarboxylic    acid butyl ester

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 8 —OH —OH H C₂₄H₂₃N₃O₄418.17 418.4

-   8.    1-{(R)-3-Biphenyl-4-yl-2-[(2-hydroxy-pyrimidine-5-carbonyl)-amino]-propyl}-cyclopropanecarboxylic    acid

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 9 —OCH₂CH₃ —COOCH₂CH₃H C₂₈H₃₁N₃O₅ 490.23 490.2

-   9.    5-[(R)-2-Biphenyl-4-yl-1-(1-ethoxycarbonyl-cyclopropylmethyl)-ethylcarbamoyl]-2H-pyrazole-3-carboxylic    acid ethyl ester

MS m/z: [M + H]⁺ Ex. R¹ R³ R⁴ Formula calcd found 10 —OH —OH HC₂₃H₂₃N₃O₄ 406.17 406.4

-   10.    1-{(R)-3-Biphenyl-4-yl-2-[(5-hydroxy-2H-pyrazole-3-carbonyl)-amino]-propyl}-cyclopropanecarboxylic    acid

Assay 1 In vitro Assays for the Quantitation of Inhibitor Potencies atHuman and Rat NEP, and Human ACE

The inhibitory activities of compounds at human and rat neprilysin (EC3.4.24.11; NEP) and human angiotensin converting enzyme (ACE) weredetermined using in vitro assays as described below.

Extraction of NEP Activity from Rat Kidneys

Rat NEP was prepared from the kidneys of adult Sprague Dawley rats.Whole kidneys were washed in cold phosphate buffered saline (PBS) andbrought up in ice-cold lysis buffer (1% Triton X-114, 150 mM NaCl, 50 mMtris(hydroxymethyl) aminomethane (Tris) pH 7.5; Bordier (1981) J. Biol.Chem. 256: 1604-1607) in a ratio of 5 mL of buffer for every gram ofkidney. Samples were homogenized on ice using a polytron hand heldtissue grinder. Homogenates were centrifuged at 1000×g in a swingingbucket rotor for 5 minutes at 3° C. The pellet was resuspended in 20 mLof ice cold lysis buffer and incubated on ice for 30 minutes. Samples(15-20 mL) were then layered onto 25 mL of ice-cold cushion buffer (6%w/v sucrose, 50 mM pH 7.5 Tris, 150 mM NaCl, 0.06%, Triton X-114),heated to 37° C. for 3-5 minutes and centrifuged at 1000×g in a swingingbucket rotor at room temperature for 3 minutes. The two upper layerswere aspirated off, leaving a viscous oily precipitate containing theenriched membrane fraction. Glycerol was added to a concentration of 50%and samples were stored at −20° C. Protein concentrations werequantitated using a BCA detection system with bovine serum albumin (BSA)as a standard.

Enzyme Inhibition Assays

Recombinant human NEP and recombinant human ACE were obtainedcommercially (R&D Systems, Minneapolis, Minn., catalog numbers 1182-ZNand 929-ZN, respectively). The fluorogenic peptide substrateMca-D-Arg-Arg-Leu-Dap-(Dnp)-OH (Medeiros et al. (1997) Braz. J. Med.Biol. Res. 30:1157-62; Anaspec, San Jose, Calif.) andAbz-Phe-Arg-Lys(Dnp)-Pro-OH (Araujo et al. (2000) Biochemistry39:8519-8525; Bachem, Torrance, Calif.) were used in the NEP and ACEassays respectively.

The assays were performed in 384-well white opaque plates at 37° C.using the fluorogenic peptide substrates at a concentration of 10 μM inAssay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethyleneglycol sorbitan monolaurate (Tween-20), 10 μM ZnSO₄; ACE: 50 mM HEPES,pH 7.5, 100 mM NaCl, 0.01% Tween-20, ZnSO₄). The respective enzymes wereused at concentrations that resulted in quantitative proteolysis of 1 μMof substrate after 20 minutes at 37° C.

Test compounds were assayed over the range of concentrations from 10 μMto 20 pM. Test compounds were added to the enzymes and incubated for 30minute at 37° C. prior to initiating the reaction by the addition ofsubstrate. Reactions were terminated after 20 minutes of incubation at37° C. by the addition of glacial acetic acid to a final concentrationof 3.6% (v/v).

Plates were read on a fluorometer with excitation and emissionwavelengths set to 320 nm and 405 nm, respectively Inhibition constantswere obtained by nonlinear regression of the data using the equation(GraphPad Software, Inc., San Diego, Calif.):ν=ν₀/[1+(I/K′)]where ν is the reaction rate, ν₀ is the uninhibited reaction rate, I isthe inhibitor concentration and K′ is the apparent inhibition constant.

Compounds of the invention were tested in this assay and found to havepK_(i) values at human NEP as follows. In general, either the prodrugcompounds did not inhibit the enzyme in this in vitro assay, or theprodrugs were not tested (n.d.) since activity would not be expected.

Ex. pK_(i) 1A n.d. 1B 8.0-8.9 2-1 7.0-7.9 2-2 7.0-7.9 2-3 7.0-7.9 2-48.0-8.9 2-5 8.0-8.9 2-6 7.0-7.9 2-7 ≧9.0 2-8 n.d. 2-9 ≧9.0  2-10 8.0-8.9 2-11 8.0-8.9  2-12 8.0-8.9  2-13 ≧9.0 3A n.d. 3B 8.0-8.9 4-1 7.0-7.94-2 6.0-6.9 4-3 6.0-6.9 4-4 7.0-7.9 4-5 7.0-7.9 4-6 8.0-8.9 4-7 7.0-7.94-8 7.0-7.9 5  8.0-8.9 6A ≧9.0 6B n.d. 6C n.d. 6D n.d. 7-1 8.0-8.9 7-28.0-8.9 7-3 ≧9.0 7-4 n.d. 7-5 n.d. 7-6 n.d. 7-7 n.d. 7-8 7.0-7.9 7-97.0-7.9  7-10 ≧9.0  7-11 ≧9.0  7-12 ≧9.0  7-13 7.0-7.9  7-14 8.0-8.9 7-15 7.0-7.9  7-16 8.0-8.9  7-17 8.0-8.9  7-18 8.0-8.9  7-19 ≧9.0  7-208.0-8.9  7-21 8.0-8.9  7-22 8.0-8.9  7-23 8.0-8.9  7-24 8.0-8.9  7-258.0-8.9  7-26 ≧9.0 8A ≧9.0 8B n.d. 8C n.d. 9-1 8.0-8.9 9-2 n.d. 9-38.0-8.9 9-4 7.0-7.9 9-5 8.0-8.9 9-6 ≧9.0 9-7 n.d. 9-8 7.0-7.9 9-97.0-7.9  9-10 7.0-7.9  9-11 8.0-8.9  9-12 7.0-7.9  9-13 7.0-7.9  9-147.0-7.9  9-15 8.0-8.9  9-16 n.d.  9-17 7.0-7.9  9-18 7.0-7.9 10A  ≧9.010B  n.d. 11-1  7.0-7.9 11-2  n.d. 11-3  8.0-8.9 11-4  n.d. 11-5  ≧9.011-6  n.d. 11-7  n.d. 11-8  8.0-8.9 11-9  n.d. 11-10 7.0-7.9 n.d. = notdetermined

Assay 2 Pharmacodynamic (PD) Assay for ACE and NEP Activity inAnesthetized Rats

Male, Sprague Dawley, normotensive rats are anesthetized with 120 mg/kg(i.p.) of inactin. Once anesthetized, the jugular vein, carotid artery(PE 50 tubing) and bladder (flared PE 50 tubing) catheters arecannulated and a tracheotomy is performed (Teflon Needle, size 14 gauge)to faciliate spontaneous respiration. The animals are then allowed a 60minute stablization period and kept continuously infused with 5 mL/kg/hof saline (0.9%) throughout, to keep them hydrated and ensure urineproduction. Body temperature is maintained throughout the experiment byuse of a heating pad. At the end of the 60 minute stabilization period,the animals are dosed intravenously (i.v.) with two doses of AngI (1.0μg/kg, for ACE inhibitor activity) at 15 minutes apart. At 15 minutespost-second dose of AngI, the animals are treated with vehicle or testcompound. Five minutes later, the animals are additionally treated witha bolus i.v. injection of atrial natriuretic peptide (ANP; 30 μg/kg).Urine collection (into pre-weighted eppendorf tubes) is startedimmediately after the ANP treatment and continued for 60 minutes. At 30and 60 minutes into urine collection, the animals are re-challenged withAngI. Blood pressure measurements are done using the Notocord system(Kalamazoo, Mich.). Urine samples are frozen at −20° C. until used forthe cGMP assay. Urine cGMP concentrations are determined by EnzymeImmuno Assay using a commercial kit (Assay Designs, Ann Arbor, Mich.,Cat. No. 901-013). Urine volume is determined gravimetrically. UrinarycGMP output is calculated as the product of urine output and urine cGMPconcentration. ACE inhibition is assessed by quantifying the %inhibition of pressor response to AngI. NEP inhibition is assessed byquantifying the potentiation of ANP-induced elevation in urinary cGMPoutput.

Assay 3 In Vivo Evaluation of Antihypertensive Effects in the ConsciousSHR Model of Hypertension

Spontaneously hypertensive rats (SHR, 14-20 weeks of age) are allowed aminimum of 48 hours acclimation upon arrival at the testing site withfree access to food and water. For blood pressure recording, theseanimals are surgically implanted with small rodent radiotransmitters(telemetry unit; DSI Models TA11PA-C40 or C50-PXT, Data Science Inc.,USA). The tip of the catheter connected to the transmitter is insertedinto the descending aorta above the iliac bifurcation and secured inplace with tissue adhesive. The transmitter is kept intraperitoneallyand secured to the abdominal wall while closing of the abdominalincision with a non-absorbable suture. The outer skin is closed withsuture and staples. The animals are allowed to recover with appropriatepost operative care. On the day of the experiment, the animals in theircages are placed on top of the telemetry receiver units to acclimate tothe testing environment and baseline recording. After at least of 2hours baseline measurement is taken, the animals are then dosed withvehicle or test compound and followed out to 24 hours post-dose bloodpressure measurement. Data is recorded continuously for the duration ofthe study using Notocord software (Kalamazoo, Mich.) and stored aselectronic digital signals. Parameters measured are blood pressure(systolic, diastolic and mean arterial pressure) and heart rate.

Assay 4 In Vivo Evaluation of Antihypertensive Effects in the ConsciousDOCA-Salt Rat Model of Hypertension

CD rats (male, adult, 200-300 grams, Charles River Laboratory, USA) areallowed a minimum of 48 hours acclimation upon arrival at the testingsite before they are placed on a high salt diet. One week after thestart of the high salt diet (8% in food or 1% NaCl in drinking water), adeoxycorticosterone acetate (DOCA) pellet (100 mg, 90 days release time,Innovative Research of America, Sarasota, Fla.) is implantedsubcutaneously and unilateral nephrectomy is performed. At this time,the animals are also surgically implanted with small rodentradiotransmitters for blood pressure measurement (see Assay 3 fordetails). The animals are allowed to recover with appropriate postoperative care. Study design, data recording, and parameters measured issimilar to that described for Assay 3.

Assay 5 In Vivo Evaluation of Antihypertensive Effects in the ConsciousDahl/SS Rat Model of Hypertension

Male, Dahl salt sensitive rats (Dahl/SS, 6-7 weeks of age from CharlesRiver Laboratory, USA) are allowed at least 48 hours of acclimation uponarrival at the testing site before they were placed on a 8% NaCl highsalt diet (TD.92012, Harlan, USA) then surgically implanted with smallrodent radiotransmitters for blood pressure measurement (see Assay 3 fordetails). The animals are allowed to recover with appropriate postoperative care. At approximately 4 to 5 weeks from the start of highsalt diet, these animals are expected to become hypertensive. Once thehypertension level is confirmed, these animals are used for the studywhile continued with the high salt diet to maintain their hypertensionlevel. Study design, data recording, and parameters measured is similarto that described in Assay 3.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

What is claimed is:
 1. A compound selected from the group consisting of:

or a salt thereof; where R¹ is selected from the group consisting of—OR⁷ and —NR⁸R⁹; R^(1P) is selected from the group consisting of —O—P³,—NHP², and —NH(O—P⁴); R^(2a) is selected from the group consisting of—OH, —OP(O)(OH)₂, and —OC(O)CH(R³⁷)NH₂, and R^(2b) is —CH₃; or R^(2a)and R^(2b) are taken together to form —CH₂—CH₂—; or R^(2a) is takentogether with R⁷ to form —OCR¹⁸R¹⁹— or is taken together with R⁸ to form—OC(O)—; Z is selected from the group consisting of —CH— and —N—; X is a—C₁₋₉heteroaryl; R³ is absent or is selected from the group consistingof H; halo; —C₀₋₅alkylene-OH; —NH₂; —C₁₋₆alkyl; —CF₃; —C₃₋₇cycloalkyl;—C₀₋₂alkylene-O—C₁₋₆alkyl; —C(O)R²⁰; —C₀₋₁alkylene-COOR²¹; —C(O)NR²²R²³;—NHC(O)R²⁴;═O; —NO₂; —C(CH₃)═N(OH); phenyl optionally substituted withone or two groups independently selected from the group consisting ofhalo, —OH, —CF₃, —OCH₃, —NHC(O)CH₃, and phenyl; naphthalenyl; pyridinyl;pyrazinyl; pyrazolyl optionally substituted with methyl; thiophenyloptionally substituted with methyl or halo; furanyl; and—CH₂-morpholinyl; and R³, when present, is attached to a carbon atom;R^(3P) is selected from the group consisting of —C₀₋₅alkylene-O—P⁴,—C₀₋₁alkylene-COO—P³, and phenyl substituted with —O—P⁴; R⁴ is absent oris selected from the group consisting of H; —OH; —C₁₋₆alkyl;—C₁₋₂alkylene-COOR³⁵; —CH₂OC(O)CH(R³⁶)NH₂; —OCH₂OC(O)CH(R³⁶)NH₂;—OCH₂OC(O)CH₃; —CH₂OP(O)(OH)₂; —CH₂CH(OH)CH₂OH;—CH[CH(CH₃)₂]—NHC(O)O—C₁₋₆alkyl; pyridinyl; and phenyl or benzyloptionally substituted with one or more groups selected from the groupconsisting of halo, —COOR³⁵, —OCH₃, —OCF₃, and —SCF₃; and R⁴, whenpresent, is attached to a carbon or nitrogen atom; R^(4P) is selectedfrom the group consisting of —O—P⁴; —C₁₋₂alkylene-COO—P³; and phenyl orbenzyl substituted with —COO—P³; or R³ and R⁴ are taken together to form-phenylene-O—(CH₂)₁₋₃- or -phenylene-O—CH₂—CHOH—CH₂—; a is 0 or 1; R⁵ isselected from the group consisting of halo, —CH₃, —CF₃, and —CN; b is 0or an integer from 1 to 3; each R⁶ is independently selected from thegroup consisting of halo, —OH, —CH₃, —OCH₃, and —CF₃; R⁷ is selectedfrom the group consisting of H; —C₁₋₈alkyl; —C₁₋₃alkylene-C₆₋₁₀aryl;—C₁₋₃alkylene-C₁₋₉heteroaryl; —C₃₋₇cycloalkyl;—[(CH₂)₂O]₁₋₃CH₃;—C₁₋₆alkylene-OC(O)R¹⁰; —C₁₋₆alkylene-NR¹²R¹³;—C₁₋₆alkylene-C(O)R³¹; —C₀₋₆alkylenemorpholinyl;—C₁₋₆alkylene-SO₂—C₁₋₆alkyl;

R¹⁰ is selected from the group consisting of —C₁₋₆alkyl, —O—C₁₋₆alkyl,—C₃₋₇cycloalkyl, —O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹²R¹³,—CH[CH(CH₃)₂]—NH₂, —CH[CH(CH₃)₂]—NHC(O)O—C₁₋₆alkyl, and—CH(NH₂)CH₂COOCH₃; and R¹² and R¹³ are independently selected from thegroup consisting of H, —C₁₋₆alkyl, and benzyl, or R¹² and R¹³ are takentogether as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—; R³¹ isselected from the group consisting of —O—C₁₋₆alkyl, —O-benzyl, and—NR¹²R^(13;) and R³² is —C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; R⁸ isselected from the group consisting of H, —OH, —OC(O)R¹⁴, —CH₂COOH,—O-benzyl, pyridyl, and —OC(S)NR¹⁵R¹⁶; R¹⁴ is selected from the groupconsisting of H, —C₁₋₆alkyl, —C₆₋₁₀aryl, —OCH₂—C₆₋₁₀aryl,—CH₂O—C₆₋₁₀aryl, and —NR¹⁵R¹⁶; and R¹⁵ and R¹⁶ are independentlyselected from the group consisting of H and —C₁₋₄alkyl; R⁹ is selectedfrom the group consisting of H, —C₁₋₆alkyl, and —C(O)R¹⁷; and R¹⁷ isselected from the group consisting of H,—C₁₋₆alkyl, —C₃₋₇cycloalkyl,—C₆₋₁₀aryl, and —C₁₋₉heteroaryl; R¹⁸ and R¹⁹ are independently selectedfrom the group consisting of H, —C₁₋₆alkyl, and —O—C₃₋₇cycloalkyl, orR¹⁸ and are taken together to form ═O; R²⁰ is selected from the groupconsisting of H and —C₁₋₆alkyl; R²¹ and R³⁵ are independently selectedfrom the group consisting of H, —C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl,—C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃,—C₁₋₆alkylene-OC(O)R²⁵, —C₁₋₆alkylene-NR²⁷R²⁸, —C₁₋₆alkylene-C(O)R³³,—C₀₋₆alkylenemorpholinyl, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

R²⁵ is selected from the group consisting of —C₁₋₆alkyl,—O—C₁₋₆alkyl,—C₃₋₇cycloalkyl, —O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR²⁷R²⁸,—CH[CH(CH₃)₂]—NH₂, —CH[CH(CH₃)₂]—NHC(O)O—C₁₋₆alkyl, and—CH(NH₂)CH₂COOCH₃; and R²⁷ and R²⁸ are independently selected from thegroup consisting of H, —C₁₋₆alkyl, and benzyl, or R²⁷ and R²⁸ are takentogether as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—; R³³ isselected from the group consisting of —O—C₁₋₆alkyl, —O-benzyl, and—NR²⁷R^(28;) and R³⁴ is —C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; R²² andR²³ are independently selected from the group consisting of H,—C₁₋₆alkyl, —CH₂COOH, —(CH₂)₂OH, —(CH₂)₂OCH₃, —(CH₂)₂SO₂NH₂,—(CH₂)₂N(CH₃)₂, —C₀₋₁alkylene-C₃₋₇cycloalkyl, and —(CH₂)₂-imidazolyl; orR²² and R²³ are taken together to form a saturated or partiallyunsaturated —C₃₋₅heterocycle optionally substituted with halo, —OH,—COOH, or —CONH₂; and optionally containing an oxygen atom in the ring;R²⁴ is selected from the group consisting of —C₁₋₆alkyl;—C₀₋₁alkylene-O—C₁₋₆alkyl; phenyl optionally substituted with halo or—OCH₃; and —C₁₋₉heteroaryl; R³⁶ is selected from the group consisting ofH, —CH(CH₃)₂, phenyl, and benzyl; and R³⁷ is selected from the groupconsisting of H, —CH(CH₃)₂, phenyl, and benzyl; where each alkyl groupin R₁, R³, and R⁴ is optionally substituted with 1 to 8 fluoro atoms;and; where the methylene linker on the biphenyl is optionallysubstituted with one or two —C₁₋₆ alkyl groups or cyclopropyl; P² is anamino-protecting group selected from the group consisting oft-butoxycarbonyl, trityl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl,formyl, trimethylsilyl, and t-butyldimethylsilyl; P³ is acarboxy-protecting group selected from the group consisting of methyl,ethyl, t-butyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl,trimethylsilyl, t-butyldimethylsilyl, and diphenylmethyl; and P⁴ is ahydroxyl-protecting group selected from the group consisting of—C₁₋₆alkyl, triC₁₋₆alkylsilyl, —C₁₋₆alkanoyl, benzoyl, benzyl,p-methoxybenzyl, 9-fluorenylmethyl, and diphenylmethyl.
 2. The compoundof claim 1, where X is selected from the group consisting of pyrazole,imidazole, triazole, benzotriazole, furan, pyrrole, tetrazole, pyrazine,thiophene, oxazole, isoxazole, thiazole, isothiazole, oxadiazole,thiadiazole, pyridazine, pyridine, pyrimidine, pyran, benzimidazole,benzoxazole, benzothiazole, pyridylimidazole, and pyridyltriazole. 3.The compound of claim 2, wherein X is selected from the group consistingof pyrazole, triazole, benzotriazole, tetrazole, oxazole, isoxazole,pyrimidine, and pyridyltriazole.
 4. The compound of claim 1, where R¹ isselected from the group consisting of —OR⁷ and —NR⁸R⁹; R⁷ is H; R⁸ is Hor —OH; and R⁹ is H.
 5. The compound of claim 1, where: R¹ is —OR⁷; andR⁷ is selected from the group consisting of —C₁₋₈alkyl,—C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl,—[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹⁰, —C₁₋₆alkylene-NR¹²R¹³,—C₁₋₆alkylene-C(O)R³¹, —C₀₋₆alkylenemorpholinyl,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

 or R¹ is —NR⁸R⁹; R⁸ is selected from the group consisting of —OC(O)R¹⁴,—CH₂COOH, —O—benzyl, pyridyl, and —OC(S)NR¹⁵R¹⁶; and R⁹ is H; or R¹ is—NR⁸R⁹; R⁸ is selected from the group consisting of —OC(O)R¹⁴, —CH₂COOH,—O—benzyl, pyridyl, and —OC(S)NR¹⁵R^(16;) and R⁹ is —C₁₋₆alkyl or—C(O)R¹⁷; or R¹ is —NR⁸R⁹; R⁸ is selected from H or —OH; and R⁹ is—C₁₋₆alkyl or —C(O)R¹⁷; or R¹ is —OR⁷ and R^(2a) is taken together withR⁷ to form —OCR¹⁸R¹⁹—; or R¹ is —NR⁸R⁹ and R^(2a) is taken together withR⁸ to form —OC(O)—.
 6. The compound of claim 1, where R¹ is —OR⁷ and R⁷is H or —C₁₋₈alkyl.
 7. The compound of claim 1, where R^(2a) is —OH andR^(2b) is —CH₃.
 8. The compound of claim 1, where R^(2a) and R^(2b) aretaken together to form —CH₂—CH₂—.
 9. The compound of claim 1, where Z is—CH—.
 10. The compound of claim 1, where Z is —N—.
 11. The compound ofclaim 1, where R³ is absent or is selected from the group consisting ofH; halo; —C₀₋₅alkylene-OH; —NH₂; —C₁₋₆alkyl; —CF₃; —C₃₋₇cycloalkyl;—C₀₋₂alkylene-O—C₁₋₆alkyl; —C(O)R²⁰; —C₀₋₁alkylene-COOR²¹; —C(O)NR²²R²³;—NHC(O)R²⁴; ═O; —NO_(2;) —C(CH₃)═N(OH); phenyl optionally substitutedwith one or two groups independently selected from the group consistingof halo, —OH, —CF₃, —OCH₃, —NHC(O)CH₃, and phenyl; naphthalenyl;pyridinyl; pyrazinyl; pyrazolyl optionally substituted with methyl;thiophenyl optionally substituted with methyl or halo; furanyl; and—CH₂-morpholinyl; and R²¹ is H.
 12. The compound of claim 1, where R³ is—C₀₋₁alkylene-COOR²¹; and R²¹ is selected from the group consisting of—C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl,—C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R²⁵,—C₁₋₆alkylene-NR²⁷R²⁸, —C₁₋₆alkylene-C(O)R³³, —C₀₋₆alkylenemorpholinyl,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,


13. The compound of claim 1, where R³ is absent or is selected from thegroup consisting of H, halo, —C₀₋₅alkylene-OH,—C₀₋₁alkylene-O—C₁₋₆alkyl, —C(O)R²⁰ ,—C₀₋₁alkylene-COOR²¹,—C(O)NR²²R²³,═O, and pyrazinyl; R²⁰ is —C₁₋₆alkyl;R²¹ is H or —C₁₋₆alkyl; R²² and R²³ are independently selected from thegroup consisting of H, —C₁₋₆alkyl, —(CH₂)₂OCH₃, and —(CH₂)₂-imidazolyl;or R²² and R²³ are taken together to form a saturated —C₃₋₅heterocycleoptionally substituted with —OH or —COOH, and optionally containing anoxygen atom in the ring.
 14. The compound of claim 1, where R⁴ is absentor is selected from the group consisting of H; —OH; —C₁₋₆alkyl;—C₁₋₂alkylene-COOR³⁵; —CH₂OC(O)CH(R³⁶)NH₂; —CH₂CH(OH)CH₂OH; pyridinyl;and phenyl or benzyl optionally substituted with one or more groupsselected from the group consisting of halo, —COOR³⁵, —OCH₃, —OCF₃, and—SCF₃; and R³⁵ is H.
 15. The compound of claim 1, where R⁴ is selectedfrom the group consisting of —OCH₂OC(O)CH₃; —CH₂OP(O)(OH)₂;—C₁₋₂alkylene-COOR³⁵; and phenyl or benzyl substituted with at least one—COOR³⁵ group; where R³⁵ is selected from the group consisting of—C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl,—C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R²⁵,—C₁₋₆alkylene-NR²⁷R²⁸,—C₁₋₆alkylene-C(O)R³³, —C₀₋₆alkylenemorpholinyl,—C₁₋₆alkylene-SO₂—C₁₋₆alkyl,


16. The compound of claim 1, where R⁴ is selected from the groupconsisting of H and —OH.
 17. The compound of claim 1, where a is
 0. 18.The compound of claim 1, where b is 0; or b is 1 and R⁶ is selected fromthe group consisting of halo, —OH, and —OCH₃; or b is 2 and each R⁶ isindependently selected from the group consisting of halo and —CH₃. 19.The compound of claim 1, where R¹ is —OR⁷; R⁷ is H or —C₁₋₈alkyl; X isselected from the group consisting of pyrazole, triazole, benzotriazole,tetrazole, oxazole, isoxazole, pyrimidine, and pyridyltriazole; R³ isabsent or is selected from the group consisting of H, halo,—C₀₋₅alkylene-OH, —C₀₋₁alkylene-O—C₁₋₆alkyl,—C(O)R²⁰—C₀₋₁alkylene-COOR²¹, —C(O)NR²²R²³;═O, and pyrazinyl; R²⁰ is—C₁₋₆alkyl; R²¹ is H or —C₁₋₆alkyl; R²² and R²³ are independentlyselected from the group consisting of H, —C₁₋₆alkyl, —(CH₂)₂OCH₃, and—(CH₂)₂-imidazolyl; or R²² and R²³ are taken together to form asaturated —C₃₋₅heterocycle optionally substituted with —OH or —COOH, andoptionally containing an oxygen atom in the ring; R⁴ is selected fromthe group consisting of H and —OH; a is 0; and b is 0; or b is 1 and R⁶is selected from the group consisting of halo, —OH, and —OCH₃; or b is 2and each R⁶ is independently selected from the group consisting of haloand —CH₃.
 20. The compound of claim 1, where R¹ is —OR⁷; R^(2a) is —OHand R^(2b) is —CH₃; or R^(2a) and R^(2b) are taken together to form—CH₂—CH₂—; X is selected from the group consisting of pyrazole,triazole, isoxazole, and pyrimidine; R³ is selected from the groupconsisting of H, —C₀₋₅alkylene-OH, and —C₀₋₁alkylene-COOR²¹; R²¹ is H or—C₁₋₆alkyl; R⁴ is selected from the group consisting of H and —OH; a is0; and b is 0; or b is 1 and R⁶ is halo; or b is 2 and each R⁶ isindependently selected from the group consisting of halo and —CH₃.